CN110997724A

CN110997724A - Methods of treating cancer using antibodies and molecules that bind BTN1A1 or BTN1A 1-ligands

Info

Publication number: CN110997724A
Application number: CN201880051242.XA
Authority: CN
Inventors: 斯蒂芬·成汉·柳; 以斯拉·明·哲·钟; 奉永植; 金龙洙
Original assignee: Stcube & Co Inc
Current assignee: Stcube & Co Inc
Priority date: 2017-06-06
Filing date: 2018-06-05
Publication date: 2020-04-10
Also published as: US20230279112A1; JP2023123451A; KR20200026209A; US11542331B2; US20200131269A1; WO2018226671A1; EP3635007A1; JP2020522562A

Abstract

The present invention provides methods of treating cancer using molecules having antigen binding fragments that immunospecifically bind to BTN1a1 or BTN1a1 ligand, such as anti-BTN 1a1 antibody or anti-BTN 1a1 ligand antibody. The invention also provides BTN1A1 ligands, such as galectin-1 (galectin-1), galectin-9 (galectin-9), neuropilin 2(neuropilin-2), and B-and T-lymphocyte attenuating proteins.

Description

Methods of treating cancer using antibodies and molecules that bind BTN1A1 or BTN1A 1-ligands

Cross Reference to Related Applications

The present application claims priority of U.S. provisional application No.62/516, 071 filed on 6/2017; the disclosure of which is incorporated herein by reference in its entirety.

Reference to sequence listing

This application was filed with a Computer Readable Form (CRF) copy of the sequence listing named 140,325-; the entire contents of which are incorporated herein by reference.

1. Field of the invention

The present invention relates generally to the fields of cancer immunology and molecular biology. The present invention provides methods of treating cancer using anti-BTN 1a1 antibodies or anti-BTN 1a 1-ligand antibodies or other molecules having an antigen binding fragment that immunospecifically binds to BTN1a1 or BTNLA1 ligand. In some embodiments, the anti-BTN 1a1 antibody or anti-BTN 1a1 ligand antibody can disrupt the BTN1a1-BTN1a1 ligand interaction. In some embodiments, the anti-BTN 1a1 ligand antibody comprises an anti-galectin 1(GAL-1) antibody, an anti-galectin 9(GAL-9) antibody, an anti-neuropilin-2 (NRP-2) antibody, or an anti-B-and T-lymphokine (BTLA) antibody.

2. Background of the invention

The immune system of humans and other mammals protects them from infection and disease. Many stimulatory and inhibitory ligands and receptors provide a tight control system that limits autoimmunity while maximizing the immune response against infection. Recently, therapeutic agents that modulate immune responses, such as anti-PD 1 or anti-PDL 1 antibodies, have been found to be effective in the treatment of certain cancers. However, there remains an urgent need to develop new therapeutic agents that safely and effectively treat diseases by modulating the immune system, particularly for resistant or refractory cancers for anti-PD 1 therapy or anti-PD-L1 therapy. The methods described herein address these needs and provide other related advantages.

3. Summary of the invention

In one aspect, the invention provides a molecule comprising an antigen binding fragment that immunospecifically binds to BTN1a1, whereby the molecule inhibits the binding of a BTN1a1 ligand, such as galectin-1 (GAL-1), galectin-9 (GAL-9), NRP-2(NRP-2), or B-and T-lymphotropic protein (BTLA), to BTN1a 1.

In some embodiments, the antigen binding fragment immunospecifically binds BTN1a1, and the molecule inhibits binding of BTN1a1 to GAL-1.

In some embodiments, the antigen binding fragment immunospecifically binds BTN1a1, and the molecule inhibits binding of BTN1a1 to GAL-9.

In some embodiments, the antigen binding fragment immunospecifically binds BTN1a1, and the molecule inhibits binding of BTN1a1 to NRP-2.

In some embodiments, the antigen binding fragment immunospecifically binds BTN1a1, and the molecule inhibits binding of BTN1a1 to BTLA.

In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1, and the molecule inhibits two or more BTN1a1 ligands, e.g., GAL-1, GAL-9, NRP-2, or BTLA.

In some embodiments, the antigen binding fragment immunospecifically binds to the extracellular domain (ECD) of BTN1a 1.

In another aspect, the invention provides a molecule comprising an antigen-binding fragment that immunospecifically binds to a BTN1a1 ligand selected from GAL-1, GAL-9, NRP-2, and BTLA, which molecule inhibits the binding of BTN1a1 ligand to BTN1a 1.

In some embodiments, the antigen-binding fragment immunospecifically binds GAL-1, and the molecule inhibits the binding of GAL-1 to BTN1a 1.

In some embodiments, the antigen-binding fragment immunospecifically binds GAL-9, and the molecule inhibits binding of GAL-9 to BTN1a 1.

In some embodiments, the antigen-binding fragment immunospecifically binds to NRP-2, and the molecule inhibits binding of NRP-2 to BTN1a 1.

In some embodiments, the antigen binding fragment immunospecifically binds BTLA, and the molecule inhibits binding of BTLA to BTN1a 1.

In some embodiments, the molecule modulates the activity or signaling of BTN1a1, or modulates the activity or signaling of a BTN1a1 and BTN1a1 ligand complex, such as GAL-1, GAL-9, NRP-2, or BTLA.

In some embodiments, the molecule inhibits binding of BTN1a1 ligand to BTN1a1 with an IC50 of no greater than 1 μ Μ.

In some embodiments, the molecule inhibits binding of a BTN1a1 ligand to BTN1a1 with an IC50 of no greater than 500nM, no greater than 400nM, no greater than 300nM, no greater than 200nM, no greater than 100nM, no greater than 50nM, no greater than 10nM, or no greater than 5 nM.

In some embodiments, the molecule can modulate T cell activity.

In some embodiments, the T cell is a CD8+ cell.

In some embodiments, the molecule can increase T cell activation or T cell proliferation.

In some embodiments, the molecule may inhibit T cell apoptosis.

In some embodiments, the antigen binding fragment preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a 1.

In some embodiments, the antigen binding fragment preferentially binds to dimeric BTN1a1 relative to monomeric BTN1a 1.

In some embodiments, the antigen-binding fragment has a dissociation constant (K) of no greater than 1 μ M_D) Immunospecifically binds to BTN1a1 or BTN1a1 ligand.

In some embodiments, the antigen-binding fragment has a dissociation constant (K) of no greater than 500nM, no greater than 400nM, no greater than 300nM, no greater than 200nM, no greater than 100nM, no greater than 50nM, no greater than 10nM, or no greater than 5nM_D) Immunospecifically binds to BTN1a1 or BTN1a1 ligand.

In some embodiments, the antigen binding fragment immunospecifically binds to K of BTN1a1 or BTN1a1 ligand_DK less than or equal to BTN1A1-GAL-1 interaction, BTN1A1-GAL-9 interaction, BTN1A1-NRP2 interaction, or BTN1A1-BTLA interaction_D。

In some embodiments, the antigen binding fragment immunospecifically binds to K of BTN1a1 or BTN1a1 ligand_DK compared to BTN1A1-GAL-1-1 interaction, BTN1A1-GAL-9 interaction, BTN1A1-NRP2 interaction, or BTN1A1-BTLA interaction_DAt least 2 times lower, at least 5 times lower, at least 10 times lower, at least 15 times lower, at least 20 times lower, at least 25 times lower, at least 30 times lower, at least 40 times lower or at least 50 times lower.

In some embodiments, inhibition of BTN1a1 binding, BTN1a1 ligand binding, or BTN1a1 ligand binding to BTN1a1 is analyzed by co-immunoprecipitation (co-IP), Surface Plasmon Resonance (SPR) assay, b-galactosidase complementation, or biolayer interferometry (BLI).

In some embodiments, the molecule is an antibody.

In some embodiments, the molecule is a monoclonal antibody.

In some embodiments, the antibody is a human or humanized antibody.

In some embodiments, the antibody is an IgG, IgM, or IgA.

In some embodiments, the molecule is a Fab ', F (ab ')2, F (ab ')3, monovalent scFv, bivalent scFv or single domain antibody.

In some embodiments, the molecule is recombinantly produced.

In another aspect, the invention provides a pharmaceutical composition comprising the provided molecule and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

In another aspect, the invention provides a method of activating a T cell, comprising contacting the T cell with an effective amount of a molecule provided herein, thereby activating the T cell by inhibiting binding of a BTN1a1 ligand to BTN1a 1.

In another aspect, the invention provides a method of inhibiting the binding of a BTN1a1 ligand to BTN1a1 expressed by a T cell, comprising contacting the T cell with an effective amount of a molecule provided herein, thereby inhibiting the binding of the BTN1a1 ligand to the T cell.

In some embodiments, the T cell is a CD8+ cell.

In some embodiments, T cell activation comprises (i) increasing T cell proliferation, (ii) decreasing T cell apoptosis, or (iii) increasing cytokine production.

In some embodiments, the cytokine is IFN gamma or IL-2.

In another aspect, the invention provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a molecule or pharmaceutical composition provided herein, wherein the molecule inhibits binding of BTN1a1 ligand to BTN1a1 in the subject.

In another aspect, the invention provides a method of inhibiting binding of BTN1a1 ligand to BTN1a1 by administering to a subject a therapeutically effective amount of a molecule or pharmaceutical composition provided herein, wherein the molecule inhibits binding of BTN1a1 ligand to BTN1a1 in the subject.

In some embodiments, the method further comprises administering high-dose radiation therapy to the patient.

In some embodiments, the cancer may include lung cancer, prostate cancer, pancreatic cancer, ovarian cancer, liver cancer, head and neck cancer, breast cancer, and gastric cancer.

In some embodiments, the cancer may comprise a lung cancer.

In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC).

In some embodiments, the NSCLC is squamous NSCLC.

In some embodiments, the cancer is an anti-PD-1 therapy or an anti-PD-L1 therapy resistant or refractory cancer.

In some embodiments, the cancer is a breast cancer or a lung cancer.

In some embodiments, the cancer is breast cancer or lewis lung cancer.

In some embodiments, the molecule is not STC810 as described in international patent application No. pct/US16/64436, or comprises a CDR or VH or VL amino acid sequence of STC 810. .

4. Brief description of the drawings

The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1-identification of galectin-1, galectin-2 and neuropilin-2 as BIN1A1 conjugates. FIG. 1 shows images of illustrative results of membrane protein array experiments again confirming galectin-1 (GAL-1, "LGALSL"), galectin-2 (GAL-2, "LGALS 9"), and neuropilin-2 (NRP-2) as exemplary properties of BTN1A1 ligands. Expression vectors for the expressed proteins were designated repeatedly on two slides ("rep 1", "rep 2") and transfected in reverse into HEK293T cells. The transfected cells were then fixed and tested with CTLA-4-Fc, BTN1A1-2NQ-Fc (non-glycosylated), BTN1A-Fc (glycosylated) or secondary antibody (cells expressing CD86/ZsGreen1 positive control vector) alone. Binding of the probe protein to the expressed protein was detected using fluorescence imaging after addition of a fluorescently labeled secondary antibody.

FIGS. 2A-C-confirmation by immunoprecipitation that GAL-1 and GAL-9 are BTN1A1 ligands. FIG. 2A shows a schematic of BTN1A1 and BTN1A 1-ligand (GAL-1 or GAL-9) protein constructs for immunoprecipitation. BTN1A1 includes an extracellular domain (ECD), a transmembrane domain (TM), a Cytoplasmic Protein Domain (CPD) and a Flag-tag. BTN1A 1-ligands included Myc-and Flag-tags. FIG. 2B shows a graph illustrating an immunoprecipitation experiment. BTN1a1 or BTN1a 1-ligand was pulled down from HEK293T cell lysate using beads coated with anti-BTN 1a1 antibody or anti-Myc antibody. FIG. 2C shows an image of a Western blot (western blots) after immunoprecipitation. Asterisks indicate bands of BTN1a1(×) or BTN1a 1-ligand (×).

FIGS. 3A-D-analysis of BTN1A1-GAL-1 interaction by Surface Plasmon Resonance (SPR). FIGS. 3A-D show sensorgrams for an exemplary SPR assay. GAL-1 protein was injected on the sensor chip with immobilized glycosylated wild-type BTN1Al-Fc (fig. 3A), non-glycosylated BTN1a1-2NQ-Fc (fig. 3B), glycosylated wild-type BTN2a1 (fig. 3C) or glycosylated wild-type BTN3A2 (fig. 3D).

FIGS. 4A and 4B-identification of BTLA as a ligand for BTN1A1 FIG. 4A shows a schematic illustrating a β -galactosidase (β -Gal) complementation assay, wherein β -Gal is divided into an Enzyme Donor (ED) and an Enzyme Acceptor (EA). interaction of the ED fusion protein with the EA fusion protein results in the reconstitution of a detectable functional β -Gal using a luminescent β -Gal substrate FIG. 4B shows a bar graph illustrating the results of an exemplary β -galactosidase (β -Gal) complementation assay.

Figure 5-validation of BTLA as BTN1a1 ligand by immunoprecipitation. FIG. 5 shows exemplary Western blot results after immunoprecipitation of BTN1A1-Flag or BTLA-Myc-Flag with anti-Myc antibody or anti-BTN 1A1 antibody (STC 810).

FIGS. 6A and 6B-analysis of BTN1A1-BTLA interaction by Surface Plasmon Resonance (SPR). FIG. 6A shows a sensorgram for SPR analysis, where GAL-1, BTLA or a control protein (control 1, control 2 or control 3) was injected at a single concentration of 3.2. mu.M onto a sensor chip immobilized with glycosylated wild-type BTN1 Al-Fc. FIG. 6B shows a sensorgram of SPR analysis in which BTLA is injected at the indicated concentrations onto a sensor chip with immobilized glycosylated wild-type BTN1A 1-Fc.

Figure 7-analysis of BTN1a1-BTLA interaction by biolayer interferometry (BLI). FIG. 7 shows a sensorgram for a BLI experiment in which soluble BTLA was contacted with immobilized BTN1A-Fc at the indicated concentrations.

5. Detailed description of the invention

The B7 family of costimulatory molecules can drive activation and suppression of immune cells. The related molecular family, the lactophilins (buryrophilins), also has immunomodulatory functions similar to those of the B7 family. Cremophil protein (Butyrophilin), subfamily 1, member a1 ("BTN 1a 1") is a type I membrane glycoprotein and is a major component of milk fat globule membrane, with structural similarity to the B7 family. BTN1a1 is known to be the major protein regulating fat droplet formation in milk. (Ogget al. PNAS,101(27): 10084-. BTN1a1 is expressed in immune cells, including T cells. Treatment with recombinant BTN1a1 was found to be an animal model that inhibited T cell activation and protected EAE. (Stefferl et al, J.Immunol.165(5):2859-65 (2000)).

BTN1a1 is also specific and highly expressed in cancer cells. BTN1a1 expressed in cancer cells is typically glycosylated. Expression of BTN1a1 can be used to aid in cancer diagnosis and to assess the effectiveness of cancer therapy.

The present disclosure is based, at least in part, on the discovery that Gal-1, Gal-9, NRP-2 and BTLA can act as BTN1A1 ligands. See, for example, examples 1 and 2. Without being bound by any particular theory, it is believed that inhibiting complex formation of GAL-1, GAL-9, NRP-2, or BTLA with BTN1a1, including disrupting the already formed complex of BTN1a1 with BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), may modulate BTLA activity or signaling. It is also believed that such modulation of BTLA activity or signaling may activate T cells (e.g., CD8+ T cells), for example, by promoting T cell proliferation, inhibiting T cell apoptosis, or inducing cytokine secretion (e.g., IFN γ or IL 2). T cell activation can result in an anti-cancer immune response that is useful for treating or preventing cancer.

The present invention provides methods of treating cancer using anti-BTN 1a1 antibodies, anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibodies, and other molecules capable of immunospecifically binding to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) and inhibiting the BTN1A-BTN1 ligand complex. Also provided are methods of diagnosing cancer and selecting patients using such anti-BTN 1a1 antibodies or anti-BTN 1a 1-ligand antibodies and other molecules capable of immunospecifically binding to BTN1a1 or BTN1a1 ligand.

5.1. Definition of

As used herein, the terms "a," "an," and "the" refer to one or more of the grammatical object of the article, unless otherwise specified. By way of example, an antibody is one antibody or more than one antibody.

As used herein, unless otherwise specified, the terms "cremophilic protein, subfamily 1, member a 1" or "BTN 1a 1" refer to any vertebrate-derived BTN1a1, including mammals such as primates (e.g., humans, cynomolgus monkeys (cyno)), dogs and rodents (e.g., mice and rats). Unless otherwise indicated, BTN1a1 also includes various BTN1a1 isoforms, related BTN1a1 polypeptides (including SNP variants thereof), and different modified forms of BTN1a1, including but not limited to phosphorylated BTN1a1, glycosylated BTN1a1, and ubiquinated BTN1a 1. Glycosylated BTN1a1 as used herein includes BTN1a1 with N55, N215, and/or N449 glycosylation.

An exemplary human BTN1a1 amino acid sequence (BC096314.1GI:64654887) is provided below, wherein potential glycosylation sites are indicated in bold and underlined:

the following provides an exemplary coding nucleic acid sequence for human BTN1a1(BC096314GI: 64654887):

an exemplary amino acid sequence of an exemplary dimeric BTN1A1 extracellular domain construct (BTN1A1-ECD-Fc) is provided below.

An exemplary amino acid sequence of an exemplary monomeric BTN1a1 extracellular domain construct (BTN1a1-His6) is provided below.

An exemplary mouse BTN1A1 amino acid sequence (GenBank: AAH11497.1) is provided below, with potential glycosylation sites indicated in bold and underlined:

an exemplary coding nucleic acid sequence for mouse BTN1A1(GenBank: BC011497.1) is provided below:

as used herein, unless otherwise specified, the terms "galectin-1", "GAL 1", "GBP" or "LGALS 1" include polypeptides ("polypeptides" and "proteins" are used interchangeably herein) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (crabs)), dogs and rodents (e.g., mice and rats), including any native polypeptide, unless otherwise specified. In certain embodiments, the term "related GAL-1 polypeptide" includes SNP variants thereof. The term "GAL-1" also includes "full-length," untreated GAL-1, and any form of GAL-1 produced by treatment in a cell. The NCBI reference sequence NP _002296 provides an exemplary human GAL-1 amino acid sequence. The NCBI reference sequence NM-002305 provides an exemplary human GAL-1 nucleic acid sequence (mRNA).

An exemplary amino acid sequence of human GAL-1(NCBI reference sequence NP-002296) is provided below.

An exemplary nucleic acid sequence for human GAL-1(NCBI reference sequence NM-002305 (coding sequence)) is provided below.

As used herein, unless otherwise specified, the terms "galectin-9", "HUAT", "GAL-9", "GAL 9", "LGALS 9" or "LGALS 9A" include polypeptides ("polypeptides" and "proteins" are used interchangeably herein) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (crabs)), dogs and rodents (e.g., mice and rats), including any native polypeptide, unless otherwise specified. In certain embodiments, the term includes "related GAL-9 polypeptides," including SNP variants thereof. The term "GAL-9" also includes "full-length," untreated GAL-9, and any form of GAL-9 produced by treatment in a cell. The NCBI reference sequence NP _001317092 provides an exemplary human GAL-9 amino acid sequence. The NCBI reference sequence NM — 002308 provides an exemplary human GAL-9 nucleic acid sequence (mRNA).

An exemplary amino acid sequence of human GAL-9(NCBI reference sequence NP-001317092) is provided below.

An exemplary nucleic acid sequence for human Gal-9(NCBI reference sequence NM-002308 (coding sequence) is provided below.

As used herein, unless otherwise specified, the terms "neuropilin-2", "NRP 2", "NP 2", "PR 02714" or "VEGF 165R 2" include polypeptides ("polypeptides" and "proteins" are used interchangeably herein) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (crabs)), dogs and rodents (e.g., mice and rats), including any native polypeptide, unless otherwise specified. In certain embodiments, the term includes "related NRP-2 polypeptides," including SNP variants thereof. The term "NRP-2" also includes "full-length," untreated NRP-2, and any form of NRP-2 produced by treatment in a cell. The NCBI reference sequence NP _003863 provides an exemplary human NRP-2 amino acid sequence. The NCBI reference sequence NM — 003872 provides an exemplary human NRP-2 nucleic acid sequence (mRNA).

An exemplary amino acid sequence of human NRP-2(NCBI reference sequence NP-003863) is provided below.

Exemplary nucleic acid sequences for human GAL-1(NCBI reference sequence NM-003872 (coding sequence)) are provided below.

As used herein, unless otherwise specified, the term "B-and T-lymphocyte attenuating protein" or "BTLA" refers to BTLA from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (crabs)), dogs, and rodents (e.g., mice and rats). Unless otherwise indicated, BTLA also includes various BTLA isoforms, related BTLA polypeptides, including SNP variants thereof, and different modified forms of BTLA, including but not limited to phosphorylated BTLA, glycosylated BTLA, and ubiquinated BTLA.

Exemplary amino acid sequences of human BTLA are provided below, in which the N-linked glycosylation sites are bold and underlined (N75, N94, and NL10):

an exemplary nucleic acid sequence of human BTLA (NCBI reference sequence NM — 001085357.1 (coding sequence)) is provided below:

as used herein, unless otherwise specified, the terms "programmed death 1", "programmed cell death 1", "protein PD-1", "PD-1 polypeptide" or "PD 1" include polypeptides (herein "polypeptide" and "protein" are used interchangeably) from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (crab-eating), dogs and rodents (e.g., mice and rats), including any native polypeptide, unless otherwise specified. In certain embodiments, the term includes "related PD-1 polypeptides," including SNP variants thereof. The term "PD-1" also includes "full-length," untreated PD-1, and any form of PD-1 that results from treatment in a cell. NCBI reference sequence NP _005009.2 provides an exemplary human PD-L1 amino acid sequence. GenBank^TMAccession number L27440.1 provides an exemplary human PD-1 nucleic acid sequence.

As used herein, unless otherwise indicated, the term "anti-PD-1 therapy" includes any PD-1 inhibitor. In some embodiments, the anti-PD-1 therapy may include an anti-PD-1 antibody or antigen-binding fragment thereof, an inhibitory nucleic acid, or a soluble PD-1 ligand (e.g., soluble PD-L1), or a fusion protein thereof (e.g., Fc-fusion protein). In some embodiments, the anti-PD-1 treatment comprises nivolumab (opdivo), pembrolizumab (Keytruda), pidilizumab (pidilizumab), AMP-514, or AMP-224.

In some embodiments, the anti-PD-1 therapy comprises nivolumab (CAS registry number 946414-94-4). Nivolumab is also known as MDX-1106, MDX-1106-04, ONO-4538 or BMS-936558. Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in US8008,449 and WO 2002/121168.

In some embodiments, the anti-PD-1 treatment comprises pembrolizumab. Pembrolizumab also known as

lambrolizumab, Merck3745, MK-3475 or SCH-900475. Pembrolizumab is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab is disclosed in, for example, Hamid, O. et al, New England Journal of medicine369(2):134-44, WO2002/114335, and US 8354509.

In some embodiments, the anti-PD-1 treatment is pidilizumab (pidilizumab). Pidizumab, also known as CT-011(CureTech), is a humanized IgG1 monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO 2002/101611.

In some embodiments, the anti-PD-1 therapy comprises the use of an anti-PD-1 antibody provided in international application PCT/US 20126/64394.

Other anti-PD 1 antibodies useful as anti-PD 1 treatments are disclosed in US8,609,089, US2010028330 and/or US 20120114649.

In some embodiments, the anti-PD-1 therapy includes the fusion protein AMP514 (amplimune). AMP-224, also known as B7-DCIg, is disclosed, for example, in WO2010/027827 and WO 2011/066342. AMP-224 is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1.

In certain embodiments, the anti-PD-1 therapy comprises an immunoadhesin (e.g., an immunoadhesin comprising an extracellular portion of PD-L1 or PD-L2, or a PD-1 binding portion fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In certain embodiments, the anti-PD-1 therapy comprises the fusion protein AMP-224 (Fc fusion of PD-L2).

As used herein, unless otherwise specified, the terms "programmed death 1 ligand 1", "programmed cell death 1 ligand 1", "protein PD-L1", "PD-L1", "PD-L1 polypeptide", or "PD 1-L1" encompass polypeptides ("polypeptides" and "proteins" are used interchangeably herein) including any native polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and macaques), dogs, and rodents (e.g., mice and rats), unless otherwise stated. In certain embodiments, the term includes "related PD-L1 polypeptides," including SNP variants thereof. Term "PD-L1 "also encompasses" full-length, "unprocessed PD-L1, as well as any form of PD-L1 that results from processing in a cell. NCBI reference sequence NP _054862.1.2 provides an exemplary human PD-L1 amino acid sequence. GenBank^TMAccession No. NM _014143 provides an exemplary human PD-1 nucleic acid sequence.

As used herein, unless otherwise specified, the term "anti-PD-L1 therapy" encompasses any inhibitor of PD-L1. In certain embodiments, the anti-PD-1 therapy can include an anti-PD-L1 antibody or antigen-binding fragment thereof, an inhibitory nucleic acid, or a soluble PD-L1 ligand (e.g., soluble PD-1), or a fusion protein thereof (e.g., Fc-fusion protein). In certain embodiments, the anti-PD-L1 therapy comprises yw243.55.s70, MPD13280A, MEDI-4736, MSB-0010718C, or MDX-1105.

In certain embodiments, the anti-PD-L1 therapy comprises MDX-1105. MDX-1105 is also known as BMS-936559. See, for example, WO 2007/005874.

In certain embodiments, the PD-L1 therapy includes the antibody yw243.55.s70, e.g., as described in WO 2010/077634 (heavy and light chain variable region sequences are shown in SEQ ID NOs: 20 and 21, respectively).

In certain embodiments, the PD-L1 therapy comprises MDPL3280A (Genentech/Roche). MDPL3280A is a human Fc optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies against PD-L1 are disclosed, for example, in U.S. patent No.7,943,743 and U.S. publication No. 20120039906.

In certain embodiments, the anti-PD-L1 therapy comprises antibody MSB0010718C (Merck Serono). MSB0010718C is also known as A09-246-2.

In certain embodiments, the anti-PD-L1 therapy comprises MDPL3280A (Genentech/Roche), a human Fc-optimized IgG1 monoclonal antibody that binds PD-L1. MDPL3280A and other human monoclonal antibodies against PD-L1 are disclosed, for example, in U.S. patent No.7,943,743 and U.S. publication No. 20120039906.

In certain embodiments, the anti-PD-L1 therapy comprises an antibody provided in international application No. pct/US2016/024691 and international application No. pct/US2017/024027 disclosed in WO 2016/160792a 1.

As used herein, unless otherwise specified, the term "Antibody" refers to the B-cell polypeptide product within the immunoglobulin (or "Ig") class of polypeptides, which are capable of binding a particular molecular antigen, and which consist of two identical pairs of polypeptide chains, such that each pair has one heavy chain (about 50-70kDa) and one light chain (about 25kDa), each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region (see borrebae (ed.) (1995) Antibody Engineering, Second Edition, oxford university press.; Kuby (1997) Immunology, Third Edition, w.h.freeman and Company, New York). Here, the specific molecular antigen includes the target BTN1a1, which may be a BTN1a1 polypeptide, a BTN1a1 fragment, or a BTN1a1 epitope. Antibodies provided herein include, but are not limited to, monoclonal antibodies, synthetic antibodies, recombinantly produced antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, endosomal (intrabodies), anti-idiotypic (anti-Id) antibodies.

As used herein, unless otherwise specified, the term "monoclonal antibody" refers to an antibody that is the product of a single cell clone or hybridoma, or a population of cells derived from a single cell. Monoclonal antibodies are also intended to refer to antibodies produced by recombinant means from immunoglobulin genes encoding the heavy and light chains, resulting in a single molecule of immunoglobulin species. The amino acid sequences of the antibodies in the monoclonal antibody preparations are substantially homogeneous, and the binding activity of the antibodies in such preparations exhibits substantially the same antigen binding activity. In contrast, polyclonal antibodies are obtained from different B cells within a population, which are a combination of immunoglobulin molecules that bind a particular antigen. Each immunoglobulin of a polyclonal antibody may bind to a different epitope of the same antigen. Methods for producing monoclonal and polyclonal Antibodies are well known in the art (Harlow and Lane., Antibodies: A Laboratory Manual Spring Harbor Laboratory Press (1989) and B borebaeck (ed.), Antibody Engineering: A Practical Guide, W.H.Freeman and Co., Publishers, New York, pp.103-120 (1991)).

As used herein, unless otherwise specified, the term "human antibody" refers to an antibody having human variable regions and/or human constant regions, or portions thereof corresponding to human germline immunoglobulin sequences. Kabat et al (1991) Sequences of proteins of Immunological Interest Fifth Edition, U.S. department of Health and human Services, NIH Publication No.91-3242, describe such human germline immunoglobulin Sequences. Herein, human antibodies may include antibodies that bind BTN1a1 and are encoded by nucleic acid sequences that are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequences.

As used herein, unless otherwise specified, the term "chimeric antibody" refers to an antibody having a portion of its heavy and/or light chain that is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain that is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No.4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA,81: 6851-.

As used herein, unless otherwise specified, the term "humanized antibody" refers to a chimeric antibody comprising a human immunoglobulin (e.g., an acceptor antibody) in which natural complementarity determining region ("CDR") residues are replaced with residues from the corresponding CDR of a non-human species (e.g., a donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In certain instances, one or more FR region residues of a human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies can have residues that are not present in the recipient antibody or the donor antibody. These modifications were made to further improve antibody performance. The humanized antibody heavy or light chain can have substantially all of at least one or more variable regions in which all or substantially all of the CDRs correspond to CDRs of a non-human immunoglobulin sequence and all or substantially all of the FRs are FRs of a human immunoglobulin sequence. The humanized antibody may have at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see Jones et al, Nature, 321:522-525 (1986); riechmann et al, Nature, 332: 323-E329 (1988); and Presta, curr, Op, struct, biol., 2: 593-; carter et al, Proc.Natl.Acd.Sci.USA89: 4285-; and U.S. Pat. nos.6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

As used herein, unless otherwise specified, the term "recombinant antibody" refers to an antibody that is prepared, expressed, created, or isolated by recombinant means. Recombinant antibodies can be antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant combinatorial antibody libraries, antibodies isolated from transgenic and/or chromosomal animals (e.g., mice or cows) (see, e.g., Taylor, l.d.et., nucleic.acids res.20:6287-6295(1992)), or antibodies prepared, expressed, created, or isolated by any other means involving splicing of immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies may have variable and constant regions, including those derived from human germline immunoglobulin Sequences (see Kabat, e.a. et al, (1991) Sequences of Proteins of immunological Interest after Edition, u.s.department of Health and human services, NIH Publication No. 91-3242). The recombinant antibodies may also be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when animals transgenic for human Ig sequences are used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies, when derived from or associated with human germline VH and VL sequences, may be sequences in the germline repertoire of human antibodies that do not naturally occur in vivo.

As used herein, unless otherwise indicated, a "neutralizing molecule" refers to a molecule that blocks BTN1a1 from binding to its natural ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, and inhibits the signaling pathway mediated by BTN1a1 and/or other physiological activities thereof. In some embodiments, the neutralizing molecule is a neutralizing antibody. In some embodiments, the neutralizing molecule comprises an antigen binding fragment that immunospecifically binds to a BTN1a1 or BTN1a1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA. IC50 for neutralizing molecules or neutralizing antibodies refers to the concentration of the molecule or antibody required to neutralize 50% of BTN1a1 in a neutralization assay. In a neutralization assay, the IC50 of the neutralizing molecule or neutralizing antibody can be in the range of 0.01-10 μ g/ml. In some embodiments, the neutralizing molecule or neutralizing antibody can immunospecifically bind BTN1a 1. In some embodiments, the neutralizing molecule or neutralizing antibody can bind to a BTN1A1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA.

As used herein, unless otherwise specified, the term "antigen-binding fragment" and similar terms refer to a portion of an antibody that includes amino acid residues that immunospecifically bind to an antigen and confer specificity and affinity of the antibody for the antigen. Antigen-binding fragments may be referred to as functional fragments of antibodies. Antigen binding fragments may be monovalent, bivalent or multivalent.

Molecules having antigen-binding fragments include, for example, Fd, Fv, Fab, F (ab '), F (ab)2, F (ab')2, single chain Fv (scFv), diabody, triabody, tetrabody, minibody, or single domain antibodies. The scFv may be a monovalent scFv or a bivalent scFv. Other molecules having an antigen-binding fragment include, for example, a heavy or light chain polypeptide, a variable region polypeptide, or a CDR polypeptide or portion thereof, so long as such antigen-binding fragment retains binding activity. Such antigen binding fragments can be found, for example, in Harlow and Lane,Antibodies:A Laboratory Manual Cold Spring Harbor Laboratory,New York(1989)；Myers(ed.),Molec.Biology and Biotechnology:A Comprehensive Desk Referencenew York, VCH publishers, Inc.; huston et al, Cell Biophysics,22: 189-; pluckthun and Skerra, meth. Enzymol,178:497-,Advanced ImmunochemistrySeconddEd., Wiley-Liss, Inc., New York, NY (1990). The antigen-binding fragment can be a peptide having at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residuesAn amino acid sequence of at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

The heavy chain of an antibody refers to a polypeptide chain of about 50-70kDa in which the amino terminal portion comprises a variable region of about 120-130 amino acids or more and the carboxy terminal portion comprises a constant region, based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types, referred to as alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ) the different heavy chains differ in size α, δ and γ contain about 450 amino acids and μ and ε contain about 550 amino acids.

A light chain of an antibody refers to a polypeptide chain of about 25kDa in which the amino terminal portion comprises a variable region of about 100 to about 110 or more amino acids, and the carboxy terminal portion comprises a constant region. The approximate length of the light chain is 211-217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called kappa (κ) and lambda (λ). Light chain amino acid sequences are well known in the art. The light chain may be a human light chain.

The variable domain or variable region of an antibody refers to a portion of the light or heavy chain of an antibody that is typically located at the amino terminus of the light or heavy chain, is about 120-130 amino acids in length in the heavy chain, is about 100-110 amino acids in the light chain, and is used for the binding and specificity of each particular antibody for its particular antigen. The variable domain sequences of different antibodies vary widely. Sequence differences are concentrated in CDRs, and the smaller variable portion in the variable domain is called the Framework Region (FR).The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with the antigen. As used herein, the numbering of amino acid positions is according to the EU index, as in Kabat et al (1991)Sequences of proteins of immunological interest(U.S. department of Health and Human Services, Washington, d.c.) 5 th edition. The variable region may be a human variable region.

CDR refers to one of the three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β -sheet framework, or one of the three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL β -sheet framework CDR is thus a variable region sequence interspersed within the framework region sequence CDR regions are well known to those skilled in the art, e.g., the region defined by Kabat et Al as the most hypervariable (V) domain of the antibody variable (V) structure (Kabat et Al, J. biol. chem.252:6609-6616 (1977); Kabat, Adv. prot. chem.32:1-75(1978)) CDR region sequences have also been structurally defined by Chothia as residues that are not part of the conserved β -sheet framework, and are therefore capable of accommodating different conformations (Chia and Lesk, J. mol. biol.196: 1997) because of these hypervariable regions are not known by comparison of the number of residues in the typical Kabat et Al, which are different in the amino acid sequences of the antibody variable region of the counterpart of the antibody variable region (Rabat positions of the amino acid sequences of the antibody variable region of the antibody V) which are known in the art (Rabat positions of the amino acid sequences of the antibody V) generally known by the amino acid sequences of.

For example, the CDRs defined according to standard nomenclature are listed in table 1 below.

TABLE 1 CDR definitions

One or more CDRs may also be incorporated covalently or non-covalently into the molecule, making it an immunoadhesin. Immunoadhesins can incorporate a CDR as part of a larger polypeptide chain, can covalently link a CDR to another polypeptide chain, or can non-covalently incorporate a CDR. The CDRs allow the immunoadhesin to bind to the specific antigen of interest.

"framework" or "FR" residues refer to those variable domain residues that flank the CDR. FR residues are present in, for example, chimeric, humanized, human domain antibodies, diabodies, linear antibodies and bispecific antibodies. FR residues are those variable region residues other than the hypervariable region residues defined herein.

As used herein, unless otherwise specified, the term "isolated" when used in reference to an antibody means that the antibody is substantially free of cellular material or other contaminating proteins from a cell or tissue source, and/or from other contaminating components from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The phrase "substantially free of cellular material" includes preparations of antibodies wherein the antibodies are isolated from cellular components of cells from which they were isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibodies having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as "contaminating protein"). In certain embodiments, when the antibody is recombinantly produced, it is substantially free of culture medium, e.g., culture medium is less than about 20%, 10%, or 5% of the volume of the protein preparation. In certain embodiments, when the antibody is produced by chemical synthesis, it is substantially free of chemical precursors or other chemicals, e.g., it is separated from chemical precursors or other chemicals involved in protein synthesis. Thus, such antibody preparations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody of interest. Contaminant components may also include, but are not limited to, materials that would interfere with the therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody will be purified (1) to greater than 95% by weight, e.g., 99% by weight, of the antibody as measured according to the Lowry method (Lowry et al J.Bio.chem.193: 265) -275,1921), (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotary cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie Brilliant blue or preferably silver yellow staining. Isolated antibodies include antibodies in situ within recombinant cells, as at least one component of the antibody's natural environment is not present. Typically, however, the isolated antibody will be prepared by at least one purification step. In particular embodiments, the antibodies provided herein are isolated.

As used herein, unless otherwise specified, the terms "polynucleotide," "nucleotide," "nucleic acid molecule," and other similar terms are used interchangeably and include DNA, RNA, mRNA, and the like.

As used herein, unless otherwise specified, the term "isolated" as applied to a nucleic acid molecule refers to a nucleic acid molecule that is separated from nucleic acid molecules that are otherwise present in a natural source. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In particular embodiments, the nucleic acid molecules encoding the antibodies provided herein are isolated or purified.

The term "binding" or "binding" as used herein refers to an interaction between molecules, unless otherwise indicated. The interaction may be, for example, a non-covalent interaction, including hydrogen bonding, ionic bonding, hydrophobic interaction, and/or van der waals interaction. The strength of the overall non-covalent interaction between an antibody and a single epitope of a target molecule such as BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA is the affinity of the antibody for the epitope. "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen).

The affinity of a binding molecule X (e.g., an antibody) for its binding partner Y can generally be determined by the dissociation constant (K)_D) And (4) showing. Low affinity antibodies generally bind antigen slowly and easilyUpon dissociation, high affinity antibodies typically bind antigen faster and tend to remain bound for longer periods of time. Various methods of measuring binding affinity are known in the art, any of which may be used for the purposes of this disclosure. ' K_D"or" K_DThe value "can be measured by assays known in the art, for example by binding assays. K_DCan be determined in a radiolabeled antigen binding assay (RIA), e.g., with the Fab type of the antibody of interest and its antigen (Chen et al (1999) J.mol.biol.293: 865-) -881). K_DOr K_DValues can also be measured by surface plasmon resonance measurements using Biacore, e.g., using Biacore M-2000 or BIacore TM-3000BIacore, Inc, Piscataway, NJ, or by bio-layer interferometry using, e.g., the OctetQK384 system (Fortebio, Menlo park, Calif.).

As used herein, unless otherwise indicated, a molecule is considered to be capable of "immunospecific binding" to a second molecule if such binding exhibits the specificity and affinity of the antibody for its associated antigen. If such binding involves the antigen recognition site of an antibody, the antibody immunospecifically binds to a target region or conformation ("epitope") of the antigen. If the other antigen has a certain sequence or conformational similarity recognized by the antigen recognition site, e.g., by immunoassay,

Antibodies that immunospecifically bind to a particular antigen can bind the other antigen with a lower affinity, as determined by assays or other assays known in the art. Antibodies typically do not bind completely unrelated antigens. Some antibodies (and antigen-binding fragments thereof) do not cross-react with other antigens. Antibodies can also bind to other molecules in a non-immunospecific manner, e.g. to FcR receptors, by virtue of a binding domain, e.g. an Fc region, in other regions/domains of the antibody not involved in the antigen recognition site.

An antibody or antigen-binding fragment that immunospecifically binds to an antigen or epitope comprising a glycosylation site can bind to both in a glycosylated form or in an unglycosylated formThe antigen or the epitope of (1). In some embodiments, the antibody or antigen binding fragment preferentially binds to a glycosylated antigen or epitope relative to a non-glycosylated antigen or epitope. The preferential binding may be determined by binding affinity. For example, an antibody or antigen-binding fragment that preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a1 may exhibit a K less than that exhibited by relatively non-glycosylated BTN1a1_DK of_DBinds glycosylated BTN1a 1. In some embodiments, the antibody or antigen binding fragment exhibits a K less than that exhibited by BTN1a1 that is relatively non-glycosylated_DK of one half_DBinds glycosylated BTN1a 1. In certain embodiments, the antibody or antigen binding fragment exhibits a K less than that exhibited by BTN1a1 that is relatively unglycosylated_DAt least 10 times K_DBinds glycosylated BTN1a 1. In some embodiments, the K to which the antibody or antigen binding fragment binds glycosylated BTN1A1_DK being non-glycosylated BTN1A1_DAbout 75%, about 50%, about 25%, about 10%, about 5%, about 2.5% or about 1%.

An antibody or antigen-binding fragment that immunospecifically binds BTN1a1 can bind to BTN1a1 monomer or BTN1a1 dimer. In some embodiments, the antibody or antigen binding fragment preferentially binds to BTN1a1 dimer relative to BTN1a1 monomer. BTN1a1 binding may occur, for example, on cell surface expressed BTN1a1 or soluble BTN1a1 domain constructs, such as BTN1a1 extracellular domain (ECD) constructs (e.g., flag-tagged BTN1a1-ECD or BTN1a1-CED-FC fusion constructs). In some embodiments, the BTN1a1 monomer or dimer is glycosylated at one or more positions. In some embodiments, the antibody or antigen binding fragment binds to K of BTN1a1 dimer_DRelative to K bound to BTN1A1 monomer_DOne half smaller. In some embodiments, the K to which the antibody or antigen binding fragment binds to BTN1A1 dimer_DRelative to K bound to BTN1A1 monomer_DAt least 10 times smaller. In some embodiments, the K to which the antibody or antigen binding fragment binds to BTN1A1 dimer_DIs K bound to ABTN1A1 monomer_DAbout 75%, about 50%, about 25%, about 10%, about 5%, about 2.5% or about 1%.

Preferential binding can also be determined by binding assays and indicated, for example, by mean fluorescence intensity ("MFI"). For example, an antibody or antigen-binding fragment that preferentially binds glycosylated BTN1a1 may bind glycosylated BTN1a1 at an MFI higher than the MFI exhibited by relatively non-glycosylated BTN1a 1. In some embodiments, the MFI that the antibody or antigen binding fragment binds to glycosylated BTN1a1 is at least two-fold greater than the MFI exhibited relative to binding to non-glycosylated BTN1a 1. In some embodiments, the MFI of the antibody or antigen binding fragment bound to glycosylated BTN1a1 is at least 3-fold greater than the MFI exhibited by binding to non-glycosylated BTN1a 1. In some embodiments, the MFI of the antibody or antigen binding fragment bound to glycosylated BTN1a1 is at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold greater than the MFI exhibited by binding to non-glycosylated BTN1a 1.

As used herein, unless otherwise specified, a molecule is said to "immunospecifically mask" glycosylation of an antigen or epitope, or its designated glycosylation site, meaning its ability to (1) block the glycosylation site of a non-glycosylated antigen or epitope, such that the antibody or epitope cannot be glycosylated, or (2) bind to the glycosylated antigen or epitope, or bind at the designated glycosylation site of the glycosylated antigen or epitope, and prevent the physiological effects of the glycosylation, e.g., downstream signal transduction mediated by the glycosylation. For example, an antibody or antigen-binding fragment that immunospecifically masks the glycosylation of BTN1a1 means that the antibody or antigen-binding fragment (1) blocks the glycosylation site of non-glycosylated BTN1a1 and prevents its glycosylation, or (2) binds to glycosylated BTN1a1 and prevents the physiological effects of the glycosylation, e.g., immunosuppression mediated by the glycosylation. As another example, an antibody or antigen-binding fragment that immunospecifically masks BTN1a1 glycosylation at N55 and N215 means that the antibody or antigen-binding fragment (1) blocks N55 and N215 of non-glycosylated BTN1a1 and prevents glycosylation at N55 and N215, or (2) binds glycosylated BTN1a1 at N55 and N215 and prevents the physiological effects of the glycosylation, e.g., immunosuppression mediated by the glycosylation.

As used herein, unless otherwise specified, the term "carrier" refers to a diluent, adjuvant (e.g., freund's adjuvant (complete or incomplete)), excipient, stabilizer, or carrier with which the therapeutic agent is administered. A "pharmaceutically acceptable carrier" is a carrier that is non-toxic to the cells or mammals to which it is exposed at the dosages and concentrations employed, and can be a sterile liquid, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.

As used herein, unless otherwise indicated, the term "vector" refers to a substance used to introduce a nucleic acid molecule into a host cell. Vectors suitable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include a selection sequence or marker operable for stable integration into the chromosome of a host cell. In addition, the vector may include one or more selectable marker genes and appropriate expression control sequences. For example, selectable marker genes may be included that provide antibiotic or toxin resistance, complement auxotrophy, or provide key nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as are well known in the art. When two or more nucleic acid molecules (e.g., antibody heavy and light chains) are to be co-expressed, both nucleic acid molecules can be inserted, for example, in a single expression vector or in different expression vectors. For single vector expression, the encoding nucleic acids may be operably linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. Introduction of a nucleic acid molecule into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blot or Polymerase Chain Reaction (PCR) amplification of mRNA, or immunoblotting for gene product expression, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. One skilled in the art will appreciate that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product (e.g., an anti-BTN 1a1 antibody provided herein), and will further appreciate that the expression level can be optimized to obtain sufficient expression using methods well known in the art.

As used herein, unless otherwise indicated, the term "host cell" refers to a particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences or integration of the nucleic acid molecule into the host cell genome that may occur in the next generation.

As used herein, unless otherwise indicated, the term "subject" refers to an animal that is the subject of treatment, observation and/or experiment. "animal" includes vertebrates and invertebrates, such as fish, shellfish, reptiles, birds, and in particular mammals. "mammal" includes, but is not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, e.g., monkeys, chimpanzees, apes, and humans.

As used herein, unless otherwise indicated, the term "cancer" or "cancerous" refers to the physiological condition of a mammal that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, hematologic cancers and solid tumors.

As used herein, unless otherwise specified, the term "treating" when used in reference to a cancer patient refers to an effect that reduces the severity of the cancer, or an effect that slows or slows the progression of the cancer, including (a) inhibiting the growth of the cancer or delaying the progression of the cancer, and (b) causing regression of the cancer, or delaying or minimizing one or more symptoms associated with the presence of the cancer.

As used herein, unless otherwise indicated, the term "resistant" or "refractory" refers to the situation where a patient has residual cancer cells (e.g., lung cancer or breast cancer cells) in a tissue or organ (e.g., lung or breast) even after intensive therapy.

The term "responsive" or "response" when used in reference to treatment refers to the degree of effectiveness of the treatment in reducing or reducing the symptoms of the disease being treated, e.g., resistant or refractory cancer to anti-PD 1 therapy or anti-PD-L1 therapy. For example, the term "increased responsiveness" as used in reference to treatment of a cell or subject refers to an increase in effectiveness in reducing or reducing the symptoms of a disease as compared to a reference treatment (e.g., of the same cell or subject, or of a different cell or subject) when measured using any method known in the art. In certain embodiments, the increase in effectiveness is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.

As used herein, the terms "effective subject response", "effective patient response" and "effective patient tumor response" refer to any improvement in the therapeutic benefit to the patient. An "effective patient tumor response" can be, for example, about 5%, about 10%, about 25%, about 50%, or about 100% reduction in the rate of tumor progression. An "effective patient tumor response" can be, for example, a reduction in the physical symptoms of cancer of about 5%, about 10%, about 25%, about 50%, or about 100%. An "effective patient tumor response" can also be, for example, an increase in patient response of about 5%, about 25%, about 50%, about 100%, about 200%, or more, as measured by any suitable means, such as gene expression, cell count, assay results, tumor size, and the like.

An improvement in cancer or cancer-related disease can be characterized as a complete response or a partial response. By "complete response" is meant no clinically detectable disease using previous abnormal radiographic studies, bone marrow and cerebrospinal fluid (CSF) or abnormal monoclonal protein measurement criteria. "partial response" refers to a reduction in measurable tumor burden (i.e., the number of malignant cells present in a subject, or the measured tumor mass volume, or the number of abnormal monoclonal proteins) in the absence of new lesions of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. The term "treatment" contemplates both a complete response and a partial response.

As used herein, the term "likelihood" generally refers to an increase in the probability of an event. When used in reference to the effectiveness of a patient's tumor response, the term "likelihood" generally expects an increase in the rate of tumor progression or the probability that tumor cell growth will be reduced. The term "likelihood" when used in reference to the effectiveness of a patient's tumor response may also generally refer to an indicator, such as an increase in mRNA or protein expression, that may demonstrate an increase in the progression of treating a tumor.

The term "predict" generally refers to determining or informing in advance. When used to "predict" the effectiveness of a cancer treatment, for example, the term "predict" can mean that the likelihood of the outcome of a cancer treatment can be determined at the beginning, either before the treatment begins, or before the treatment cycle substantially progresses.

As used herein, the term "monitoring" generally refers to the observation, supervision, regulation, monitoring, tracking, or monitoring of activity. For example, the term "monitoring the effectiveness of a compound" refers to tracking the effectiveness of a treatment for cancer in a patient or in tumor cell culture. Similarly, the term "monitoring" when used in connection with patient compliance, either individually or in clinical trials, refers to tracking or confirming that the patient actually took the drug being tested as prescribed. For example, monitoring can be performed by tracking the expression of mRNA or protein biomarkers.

The term "tumor" as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. As used herein, "neoplastic" refers to any form of abnormally regulated or unregulated cell growth, whether malignant or benign, resulting in abnormal tissue growth. Thus, "neoplastic cells" include malignant and benign cells with abnormally regulated or unregulated cell growth.

As used herein, unless otherwise specified, the term "therapeutically effective amount" refers to an amount of an agent (e.g., an antibody described herein or any other agent described herein) sufficient to reduce and/or improve the severity and/or duration of a given disease, disorder or condition and/or symptoms associated therewith. A therapeutically effective amount of an agent comprising a therapeutic agent can be an amount necessary to (1) reduce or ameliorate the progression or development of a given disease, disorder or condition, (ii) reduce or ameliorate the recurrence, development or onset of a given disease, disorder or condition, and/or (iii) improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than administration of an antibody provided herein). The therapeutically effective amount of a substance/molecule/agent (e.g., an anti-BTN 1a1 antibody) disclosed herein may vary depending on factors such as the disease state, age, sex, and weight of the individual, the ability of the substance/molecule/agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount wherein any toxic or adverse effects of the substance/molecule/agent do not exceed a therapeutically beneficial effect.

As used herein, unless otherwise specified, the term "administering" refers to the act of injecting or physically delivering a substance present outside the body into a patient, e.g., by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other physical delivery method described herein or known in the art. When a disease, disorder or condition, or symptoms thereof, are to be treated, administration of the substance is generally carried out after the onset of the disease, disorder or condition, or symptoms thereof. Where a disease, disorder or condition, or symptoms thereof, are prevented, administration of the substance is generally carried out prior to the onset of the disease, disorder or condition, or symptoms thereof.

A "biomarker" or "biomarker" is a substance whose detection indicates a particular biological state, e.g., the presence of cancer. In certain embodiments, the biomarkers can be determined individually. In other embodiments, several biomarkers may be measured simultaneously. In certain embodiments of the methods provided herein, BTN1a1 is a biomarker indicative of the presence of cancer. In certain embodiments, PD-L1 is a biomarker indicative of the presence of cancer. In certain embodiments, BTN1a1 and PD-L1 can be used in combination to indicate the presence of a cancer (e.g., an anti-PD 1 or anti-PD-L1 therapy resistant or refractory cancer that is responsive to treatment with, for example, an anti-BTN 1a1 antibody).

In some embodiments, a "biomarker" indicates an alteration in mRNA expression levels, which may be associated with the risk or progression of a disease, or with a susceptibility to a given treatment. In some embodiments, the biomarker is a nucleic acid, such as mRNA or cDNA (e.g., BTN1a1 or PD-L1mRNA or cDNA).

In further embodiments, a "biomarker" means an alteration in the level of expression of a polypeptide or protein, which may be associated with the risk or progression of a disease, or susceptibility of a patient to treatment. In some embodiments, the biomarker may be a polypeptide or protein, or a fragment thereof (e.g., BTN1a1 or PD-L1 protein). The relative levels of a particular protein can be determined by methods known in the art. For example, antibody-based methods such as immunoblotting, enzyme-linked immunosorbent assay (ELISA) or other methods can be used.

The term "expressed" or "expression" as used herein refers to the transcription of a gene into an RNA nucleic acid molecule that is at least partially complementary to one of the two nucleic acid strands of the gene. The term "expressed" or "expression" as used herein also refers to the translation of a protein, polypeptide or portion thereof from an RNA molecule.

The term "level" refers to the amount, accumulation or rate of a biomarker molecule (e.g., BTN1a1 or PD-L1). The level may be indicated, for example, by the amount or rate of synthesis of messenger rna (mrna) encoded by the gene, the amount or rate of synthesis of a polypeptide or protein encoded by the gene, or the amount or rate of synthesis of biomolecules accumulated in the cell or biological fluid. The term "level" refers to the absolute amount of a molecule or the relative amount of a molecule in a sample determined under steady-state or non-steady-state conditions.

The terms "determining," "measuring," "evaluating," "assessing," and "analyzing," as used herein, generally refer to any form of measurement and includes determining whether an element is present. These terms include quantitative and/or qualitative determinations. The evaluation may be relative or absolute. Evaluating whether there is may include determining the amount of what is present and determining whether it is present or absent.

The term "sample" as used herein relates to a material or mixture of materials that generally comprises one or more components of interest in liquid form.

As used herein, "biological sample" refers to a sample obtained from a biological subject, including a sample of biological tissue or fluid origin obtained or collected in vivo or in situ. Biological samples also include samples from portions of a biological subject containing pre-cancerous or cancerous cells or tissues. These samples can be, but are not limited to, organs, tissues and cells isolated from mammals. Exemplary biological samples include, but are not limited to, cell lysates, cell cultures, cell lines, tissues, oral tissues, gastrointestinal tissues, organs, organelles, biological fluids, blood samples, urine samples, skin samples, and the like. Preferred biological samples include, but are not limited to, whole blood, partially purified blood, PBMC, tissue biopsy, and the like.

5.2 molecules with antigen-binding fragments that immunospecifically bind BTN1A1 or BTN1A1 ligand

Provided herein are molecules having an antigen binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, whereby the molecule is capable of inhibiting the binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the molecule can inhibit the formation of a BTN1a1-BTN1a1 ligand complex (e.g., a complex comprising GAL-1, GAL-9, NRP-2, or BTLA and BTN1a 1). In some embodiments, the molecule can disrupt the BTN1a1-BTN1a1 ligand complex formed (e.g., a complex comprising GAL-1, GAL-9, NRP-2, or BTLA and BTN1a 1). In some embodiments, the molecule is an antibody, including an anti-BTN 1A1 antibody, an anti-GAL-1 antibody, an anti-GAL-9 antibody, an anti-NRP-2 antibody, or an anti-BTLA antibody. In some embodiments, an antigen binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) binds to a fragment or epitope of BTN1a1 or BTN1a1 ligand. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 dimer. In some embodiments, the BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA epitope can be a linear epitope. In some embodiments, the BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA epitope can be a conformational epitope. In some embodiments, the BTN1a1 epitope is found in BTN1a1 dimer, but not in BTN1a1 monomer. In some embodiments, molecules provided herein having an antigen binding fragment that immunospecifically binds to BTN1a1, GAL-1, GAL-9, NRP-2, or BTLA inhibit the immunosuppressive function of BTN1a1 or BTN1a1-BTN1a1 ligand complex.

In some embodiments, the molecule is an anti-glycosylated BTN1a1 antibody or an antigen binding fragment comprising an anti-glycosylated BTN1a1 antibody. In some embodiments, the molecule is an anti-BTN 1a1 dimer antibody, or an antigen-binding fragment comprising an anti-BTN 1a1 dimer antibody. In some embodiments, the molecule is a molecule having an antigen-binding fragment that immunospecifically binds BTN1a1, as described, for example, in international patent application PCT/US20106/064436 (filed on 1/12/2016), U.S. provisional application 62/513389 (filed on 21/5/2017), or U.S. provisional application No.62/513393 (filed on 21/5/2017), the entire contents of which are incorporated herein by reference. In some embodiments, the molecule comprises an antigen binding fragment or VH, VL, or CDR sequence of anti-BTN 1a1 antibody STC703, STC810, STC820, STC1011, STC1012, or STC1029, or humanized variants thereof, as described in U.S. international patent application PCT/US20106/064436, U.S. provisional application No.62/513389, or U.S. provisional application No. 62/513393.

In some embodiments, the molecule is not STC810 or an antigen-binding fragment thereof or a VH, VL, or CDR amino acid sequence of STC810 as described in international patent application No. pct/US 20126/64436.

In some embodiments, the molecule is an anti-GAL-1 antibody or an antigen-binding fragment comprising an anti-GAL-1 antibody. In some embodiments, the molecule is an anti-GAL-1 antibody or antigen-binding fragment comprising an anti-GAL-1 antibody as described in International patent application PCT/US1974/047783 (publication WO20/013388A3), the entire disclosure of which is incorporated herein by reference.

In some embodiments, the molecule is an anti-GAL-9 antibody or an antigen-binding fragment comprising an anti-GAL-9 antibody. In some embodiments, the molecule is an anti-Gal-9 antibody or antigen-binding fragment including an anti-Gal-9 antibody as described in international patent application PCT/FR20/051498 (e.g., published as WO20125/185875a2), which is incorporated herein by reference in its entirety.

In some embodiments, the molecule is an anti-NRP-2 antibody or antigen-binding fragment including an anti-NRP-2 antibody. In some embodiments, the molecule is an anti-NRP-2 antibody or antigen-binding fragment including an anti-NRP-2 antibody as described in International patent application PCT/US2002/069179 (e.g., published as WO2002/143665Al), the entire contents of which are incorporated herein by reference.

In some embodiments, the molecule is an anti-NRP-2 antibody or antigen-binding fragment including an anti-NRP-2 antibody. In some embodiments, the molecule is an anti-NRP-2 antibody, or an antigen-binding fragment including the anti-NRP-2 antibody, for example in international patent application No. PCT/US2002/069179 (e.g., disclosed as WO2002/143665Al) or PCT/US2007069185 (e.g., disclosed as WO2002/143666a2), the entire contents of which are incorporated herein by reference.

In some embodiments, the molecule is an anti-BTLA antibody or an antigen-binding fragment including an anti-BTLA antibody. In some embodiments, the molecule is an anti-BTLA antibody or antigen-binding fragment including an anti-BTLA antibody as described in international patent application PCT/US20126/64385 (filed on 21/12/1976), PCT/US2010/043182 (e.g., disclosed as WO2011/014438, etc.), PCT/EP2010/053356 (e.g., disclosed as WO2010/106051Al), PCT/US2002/084792 (e.g., disclosed as WO1988/076560a2), the entire contents of which are incorporated herein by reference.

In some embodiments, the molecule is an anti-BTN 1a1 antibody or an antigen-binding fragment including an anti-BTN 1a1 antibody. In some embodiments, the molecule is an anti-BTN 1a1 antibody or antigen-binding fragment including an anti-BTN 1a1 antibody as described in international patent application PCT/US20106/064436 (filed on 21/12/1976), the entire contents of which are incorporated herein by reference.

In one aspect, provided herein is a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1, whereby the molecule can inhibit the binding of a BTN1a1 ligand, such as galectin-1 (GAL-1), galectin-9 (GAL-9), NRP-2(NRP-2), or B-and T-lymphocyte attenuating protein (BTLA), to BTN1a 1. In some embodiments, the molecule can inhibit binding of BTN1a1 to GAL-1. In some embodiments, the molecule may inhibit binding of BTN1a1 to GAL-9. In some embodiments, the molecule can inhibit binding of BTN1A1 to NRP-2. In some embodiments, the molecule can inhibit binding of BTN1a1 to BTLA. In some embodiments, the molecule may completely inhibit the binding of a BTN1A1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, to BTN1A 1. In some embodiments, the molecule may at least partially inhibit binding of a BTN1A1 ligand, such as Gal-1, Gal-9, NRP-2, or BTLA, to BTN1A 1. In some embodiments, the molecule can inhibit at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of a BTN1a1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, to BTN1a 1. In some embodiments, the binding of BTN1a1 to BTN1a1 ligand or its inhibition is determined using surface plasmon resonance, biolayer interferometry, or co-immunoprecipitation. In some embodiments, the molecule is capable of inhibiting the binding of a BTN1a1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, to BTN1a1 with an IC50 value of less than 1 μm, less than 900nM, less than 800nM, less than 700nM, less than 600nM, less than 500nM, less than 400nM, less than 300nM, less than 200nM, less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 60nM, less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, or less than 1 nM. In some embodiments, the neutralization assay is a surface plasmon resonance, biolayer interferometry, co-immunoprecipitation, FRET or TR-FRET assay, or ELISA.

In some embodiments, the molecule comprises an antigen binding fragment that immunospecifically binds to BTN1a1, whereby the molecule can inhibit the binding of two or more BN1a1 ligands, such as GAL-1, GAL-9, NRP-2, or BTLA, to BTN1a 1. In some embodiments, the molecule may inhibit the binding of GAL-1 and GAL-9 to BTN1a 1. In some embodiments, the molecule can inhibit the binding of GAL-1 and NRP-2 to BTN1A 1. In some embodiments, the molecule can inhibit binding of GAL-1 and BTLA to BTN1a 1. In some embodiments, the molecule can inhibit the binding of GAL-9 and NRP-2 to BTN1A 1. In some embodiments, the molecule can inhibit binding of GAL-9 and BTLA to BTN1a 1. In some embodiments, the molecule may inhibit binding of NRP-2 and BTLA to BTN1a 1. In some embodiments, the molecule may inhibit the binding of GAL-1, GAL-9 and NRP-2 to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and BTLA to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-1, NRP-2, and BTLA to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-9, NRP-2, and BTLA to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, NRP-2, and BTLA to BTN1A 1.

In some embodiments, the molecule comprises an antigen binding domain that binds to the extracellular domain (ECD) of BTN1a 1.

In another aspect, a molecule provided herein includes an antigen binding fragment that immunospecifically binds to a BTN1a1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, whereby the molecule is capable of inhibiting the binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the antigen-binding fragment immunospecifically binds to GAL-1, and the molecule is capable of inhibiting the binding of GAL-1 to BTN1a 1. In some embodiments, the antigen-binding fragment immunospecifically binds to GAL-9, and the molecule is capable of inhibiting binding of GAL-9 to BTN1a 1. In some embodiments, the antigen-binding fragment immunospecifically binds to NRP-2, and the molecule can inhibit binding of NRP-2 to BTN1A 1. In some embodiments, the antigen-binding fragment immunospecifically binds BTLA, and the molecule can inhibit binding of BTLA to BTN1a 1. In some embodiments, the molecule may completely inhibit the binding of a BTN1a1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, to BTN1a 1. In some embodiments, the molecule may at least partially inhibit binding of a BTN1a1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, to BTN1a 1. In some embodiments, the molecule can inhibit at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of a BTN1a1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, to BTN1a 1. In some embodiments, binding of BTN1a1 to BTN1a1 ligand or inhibition thereof is determined using surface plasmon resonance, biolayer interferometry, or co-immunoprecipitation. In some embodiments, the molecule is capable of inhibiting the binding of a BTN1a1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, to BTN1a1 with an IC50 value of less than 1 μ Μ, less than 900nM, less than 800nM, less than 700nM, less than 600nM, less than 500nM, less than 400nM, less than 300nM, less than 200nM, less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 60nM, less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, or less than 1 nM. In some embodiments, the neutralization assay is a surface plasmon resonance, biolayer interferometry, co-immunoprecipitation, FRET or TR-FRET assay, or ELISA.

In some embodiments, an antigen-binding fragment of a molecule provided herein can bind a BTN1a1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA, with a dissociation constant of 1 μ Μ or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less. In some embodiments, the antigen-binding fragment can bind to GAL-1 with a dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less. In some embodiments, an antigen-binding fragment can bind to Gal-9 with a dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1 μ M or less. In some embodiments, an antigen-binding fragment may bind to NRP-2 with a binding dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less. In some embodiments, an antigen-binding fragment can bind to BTLA with a binding dissociation constant of 1 μ Μ or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less.

In some embodiments, the molecule may modulate the activity or signaling of BTN1a1, or modulate the activity or signaling of a complex of BTN1a1 and BTN1a1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA).

In some embodiments, the molecule can modulate T cell activity. In some embodiments, the T cell is a CD8+ cell. In some embodiments, the molecule can increase T cell activation or T cell proliferation. In some embodiments, the molecule may inhibit T cell apoptosis.

N-glycosylation is a post-translational modification initiated in the Endoplasmic Reticulum (ER) and subsequently processed in the Golgi (Schwarz and Aebi, curr. Opin. struc. Bio., 21(5):576-582 (2011)). This type of modification is first catalyzed by the membrane-associated Oligosaccharyltransferase (OST) complex, which transfers preformed glycans composed of oligosaccharides to asparagine (Asn) side chain receptors located within the NXT motif (-Asn-X-Ser/Thr-) (Cheang and Reithmeier, Methods, 41:451-459, 2007); helenius and Aebi, Science, 291(5512):2364-9 (2001). The addition or removal of sugars from preformed glycans is mediated by a set of glycosyltransferases and glycosidases, respectively, that tightly regulate the N-glycosylation cascade in a cell-and location-dependent manner.

In some embodiments, the molecule has an antigen binding fragment that selectively binds to one or more glycosylation motifs of BTN1a 1. In some embodiments, the antigen binding fragment immunospecifically binds to a glycopeptide having a glycosylation motif and to an adjacent peptide. In some embodiments, the antigen-binding fragment immunospecifically binds to a peptide sequence that is located in three dimensions near one or more glycosylation motifs. In some embodiments, the antigen binding fragment selectively binds to one or more glycosylation motifs of BTN1a1 dimer relative to one or more glycosylation motifs of BTN1a1 monomer.

In some embodiments, the antigen binding fragment binds to glycosylated BTN1a1 (e.g., glycosylated BTN1a1 dimer) relative to K bound to non-glycosylated BTN1a1_D(ii) has a KD of less than at least 30%, 40%, 50%, 60%, 70%, 80% or 90%. In certain embodiments, the antigen binding fragment binds to glycosylated BTN1a1 relative to K bound to non-glycosylated BTN1a1_DOf which K is_DLess than 50%. In some embodiments, the antigen binding fragment binds to K of glycosylated BTN1A1_DRelative to K bound to unglycosylated BTN1A1 _D1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% smaller. In some casesIn embodiments, the antigen binding fragment binds to K of glycosylated BTN1A1_DRelative to K bound to unglycosylated BTN1A1_DAt least 10 times smaller.

The specific glycosylation site of a particular BTN1a1 isoform or variant may differ from that of the particular BTN1a1 isoform or variant at amino acid positions 55,215 or 449. In these cases, one of ordinary skill in the art would be able to determine the glycosylation site corresponding to any particular BTN1a1 isoform or variant of the above-exemplified N55, N215 and N449 of human BTN1a1 based on sequence alignment and other general knowledge in the art. Likewise, also provided herein are molecules having an antigen binding fragment that immunospecifically binds to a glycosylated form of BTN1a1 isoform or variant relative to a non-glycosylated BTN1a1 isoform or variant. The glycosylation sites for BTN1a1 isoforms or variants may be the corresponding sites for N55, N215, and N449 of the human BTN1a1 sequence provided above.

In some embodiments, the molecule has an antigen-binding fragment that immunospecifically binds to glycosylated BTN1a1 (e.g., glycosylated BTN1a1 dimer). In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at positions N55, N215, and/or N449. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at position N55. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at position N215. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at position N449. In some embodiments, the antigen-binding fragment immunospecifically binds to one or more glycosylation motifs. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at positions N55 and N215. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at positions N215 and N449. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at positions N55 and N449. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at positions N55, N215 and N449.

In some embodiments, the molecule has an antigen-binding fragment that immunospecifically binds to glycosylated BTN1a1, whereby the antigen-binding fragment preferentially binds to glycosylated BTN1a1 relative to non-glycosylated BTN1a1 (e.g., glycosylated BTN1a1 dimer). In some embodiments, the antigen binding fragment preferentially binds BTN1a1 glycosylated at positions N55, N215, and/or N449 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds BTN1a1 glycosylated at the N55 position relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 glycosylated at N215 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 glycosylated at position N449 over non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to one or more glycosylation motifs. In some embodiments, the antigen binding fragment preferentially binds BTN1a1 glycosylated at positions N55 and N215 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 glycosylated at positions N215 and N449 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds BTN1a1 glycosylated at positions N55 and N449 over non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds BTN1a1 glycosylated at positions N55, N215, and N449 relative to non-glycosylated BTN1a 1.

Preferential binding can be determined by binding affinity. For example, an antibody or antigen-binding fragment that preferentially binds to glycosylated BTN1A (e.g., glycosylated BTN1A dimer) may exhibit a K relative to the K exhibited by non-glycosylated BTN1A_DSmaller K_DCombined with glycosylated BTN 1A. In some embodiments, the antibody or antigen binding fragment binds to a glycosylated BTN1A_DRelative to K bound to non-glycosylated BTN1A_DOne half smaller. In some embodiments, the antibody or antigen binding fragment binds to K that is glycosylated BTN1A_DRelative to K bound by non-glycosylated BTN1A_DAt least 2 times smaller. In some embodiments, theAntibodies or antigen binding fragments bind to glycosylated BTN1A, K_DRatio versus K exhibited for non-glycosylated BTN1A_DAt least 5 times smaller. In some embodiments, the antibody or antigen binding fragment binds to glycosylated BTN1A, K_DRatio versus K exhibited for non-glycosylated BTN1A_DAt least 10 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K that is glycosylated BTN1A_DRelative to K with unglycosylated BTN1A_DAt least 15 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K that is glycosylated BTN1A_DRelative to K with unglycosylated BTN1A_DAt least 20 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K that is glycosylated BTN1A_DRelative to K bound to unglycosylated BTN1A_DAt least 25 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K that is glycosylated BTN1A_DRatios versus K exhibited in combination with non-glycosylated BTN1A_DAt least 30 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K that is glycosylated BTN1A_DRelative to K binding to unglycosylated BTN1A_DAt least 40 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K that binds to glycosylated BTN1A_DRelative to K bound to unglycosylated BTN1A_DAt least 50 times smaller. In some embodiments, the antibody or antigen binding fragment binds to a glycosylated BTN1A_DAbout K bound to non-glycosylated BTN1A_D75% of the total. In some embodiments, the antibody or antigen binding fragment binds to a glycosylated BTN1A_DAbout K bound to non-glycosylated BTN1A_D50% of the total. In some embodiments, the antibody or antigen binding fragment binds to a glycosylated BTN1A_DAbout K bound to non-glycosylated BTN1A_D25% of the total. In some embodiments, the antibody or antigen binding fragment binds to a glycosylated BTN1A_DAbout K bound to non-glycosylated BTN1A_D10% of the total. In some embodiments, the antibody or antigen binding fragment is conjugated to glycosylationBTN1A bound K_DAbout K bound to non-glycosylated BTN1A_D5% of the total. In some embodiments, the antibody or antigen binding fragment binds to a glycosylated BTN1A_DAbout K bound to non-glycosylated BTN1A_D2.5% of the total. In some embodiments, the antibody or antigen binding fragment binds to K bound by glycosylated BTN1A_DAbout K bound to non-glycosylated BTN1A _D1% of the total.

Preferential binding can also be determined by binding assays, as indicated by fluorescence intensity ("MFI"). For example, an antibody or antigen-binding fragment that preferentially binds to glycosylated BTN1a1 (e.g., glycosylated BTN1A dimer) exhibits a higher MFI in binding to glycosylated BTN1A relative to the MFI exhibited in binding to non-glycosylated BTN 1A. In some embodiments, the MFI that the antibody or antigen binding fragment binds to glycosylated BTN1A is at least twice as high as the MFI of non-glycosylated BTN 1A. In some embodiments, the MFI to which the antibody or antigen binding fragment binds glycosylated BTN1A is at least twice that of non-glycosylated BTN 1A. In some embodiments, the MFI that the antibody or antigen binding fragment binds to glycosylated BTN1A is at least 3-fold greater than the MFI exhibited by binding to non-glycosylated BTN 1A. In some embodiments, the MFI that the antibody or antigen binding fragment binds to glycosylated BTN1A is at least 5-fold greater than the MFI exhibited by binding to non-glycosylated BTN 1A. In some embodiments, the MFI that the antibody or antigen binding fragment binds to glycosylated BTN1A is at least 10-fold greater than the MFI exhibited by binding to non-glycosylated BTN 1A. In some embodiments, the MFI that the antibody or antigen binding fragment binds to glycosylated BTN1A is at least 15-fold greater than the MFI exhibited by binding to non-glycosylated BTN 1A. In some embodiments, the MFI that the antibody or antigen binding fragment binds to glycosylated BTN1A is at least 20-fold greater than the MFI exhibited by binding to non-glycosylated BTN 1A. In some embodiments, the antibody or antigen binding fragment binds to glycosylated BTN1A at least 25-fold the MFI exhibited by binding to non-glycosylated BTN 1A. In some embodiments, the MFI that the antibody or antigen binding fragment binds to glycosylated BTN1A is at least 30-fold greater than the MFI exhibited by binding to non-glycosylated BTN 1A. In some embodiments, the MFI that the antibody or antigen binding fragment binds to glycosylated BTN1A is at least 40-fold greater than the MFI exhibited by binding to non-glycosylated BTN 1A. In some embodiments, the MFI that the antibody or antigen binding fragment binds to glycosylated BTN1A is at least 50-fold greater than the MFI exhibited by binding to non-glycosylated BTN 1A.

In some embodiments, the antigen binding fragment immunospecifically masks BTN1A glycosylation (i.e., for example, glycosylated BTN1A dimer at positions N55, N215, and/or N449). In some embodiments, the antigen binding fragment immunospecifically masks glycosylation at position N55 of BTN 1A. In some embodiments, the antigen binding fragment immunospecifically masks glycosylation at position N215 of BTN 1A. In some embodiments, the antigen-binding fragment immunospecifically masks glycosylation at position N449 of BTN 1A. In some embodiments, the antigen binding fragment immunospecifically masks one or more glycosylation motifs of BTN 1A. In some embodiments, the antigen binding fragment immunospecifically masks glycosylation at positions N55 and N215 of BTN 1A. In some embodiments, the antigen-binding fragment immunospecifically masks glycosylation at positions N215 and N449 of BTN 1A. In some embodiments, the antigen binding fragment immunospecifically masks glycosylation at positions N55 and N449 of BTN 1A. In some embodiments, the antigen-binding fragment immunospecifically masks glycosylation at positions N55, N215 and N449 of BTN 1A.

In some embodiments, the molecule has an antigen-binding fragment that selectively binds to BTN1a1 dimer relative to BTN1a1 monomer. In some embodiments, the BTN1a1 dimer is expressed on the surface of a cell. In some embodiments, the BTN1a1 dimer is a soluble protein fragment of BTN1a1, such as an extracellular domain construct of BTN1a1, e.g., an Fc-fusion protein construct (e.g., BTN1a 1-ECD-Fc). In some embodiments, the BTN1a1 monomer is an extracellular domain construct of BTN1a1, such as a flag-tagged or His 6-tagged BTN1a1-ECD construct. In some embodiments, the molecule that selectively binds to BTN1a1 dimer is a molecule that selectively binds to glycosylated BTN1a1 provided herein. In some embodiments, preferential binding to BTN1a1 dimer over BTN1a1 monomer is determined by using, for example, surface plasmon resonance analysis (e.g., Biacore) to determine preferential binding to BTN1a1-ECD-Fc construct over BTN1a1-ECD-His6 or BTN1a1-ECD-Flag construct.

In some embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRelative to K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 30%, 40%, 50%, 60%, 70%, 80%, or 90% less. In certain embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DK relative to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_D50% smaller. In some embodiments, the antigen binding fragment binds to K of BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DK relative to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_D1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50% smaller. In some embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRelative to K with BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 10 times smaller.

Preferential binding can be determined by binding affinity. For example, an antibody or antigen-binding fragment that preferentially binds to a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer) binds to K of a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer)_DHas a small KD relative to that of binding to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer). In some embodiments, the antibody or antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRelative to K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DOne half smaller. In some embodiments, the antibody or antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRelative to K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 2 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRelative to K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 5 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRelative to K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 10 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRelative to K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 15 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRelative to K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 20 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRelative to K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 25 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRelative to K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 30 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRelative to K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 40 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRelative to K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 50 times smaller. In some embodiments, the antibody or antigen binding fragment binds to K of a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer)_DAbout alone with respect to binding BTN1A1K of body (e.g., glycosylated BTN1A1 monomer)_D75% of the total. In some embodiments, the antibody or antigen binding fragment binds to K of a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer)_DAbout relative to the K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_D50% of the total. In some embodiments, the antibody or antigen binding fragment binds to K of a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer)_DAbout relative to the K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_D25% of the total. In some embodiments, the antibody or antigen binding fragment binds to K of a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer)_DAbout relative to the K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_D10% of the total. In some embodiments, the antibody or antigen binding fragment binds to K of a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer)_DAbout relative to the K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_D5% of the total. In some embodiments, the antibody or antigen binding fragment binds to K of a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer)_DAbout relative to the K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_D1% of the total.

Preferential binding can also be determined by binding assays, as indicated by fluorescence intensity ("MFI"). For example, an antibody or antigen-binding fragment that preferentially binds to a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer) binds to a BTN1a1 monomer (e.g., a glycosylated BTN1a1 monomer) at a higher MFI than that exhibited relative to binding to a BTN1a1 monomer. In some embodiments, the antibody or antigen binding fragment binds to a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer) at least twice the MFI exhibited relative to binding to a BTN1a1 monomer (e.g., a glycosylated BTN1a1 monomer). In some embodiments, the above-described antibodies or antigen binding fragments bind to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) at least three times the MFI exhibited relative to binding to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer). In some embodiments, the above-described antibodies or antigen binding fragments bind to a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer) at least 5-fold greater than the MFI exhibited relative to binding to a BTN1a1 monomer (e.g., a glycosylated BTN1a1 monomer). In some embodiments, the above-described antibodies or antigen binding fragments bind to a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer) at least 10-fold greater MFI than that exhibited relative to binding to a BTN1a1 monomer (e.g., a glycosylated BTN1a1 monomer). In some embodiments, the above-described antibodies or antigen binding fragments bind to a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer) at least 15-fold more MFI than is exhibited relative to binding to a BTN1a1 monomer (e.g., a glycosylated BTN1a1 monomer). In some embodiments, the antibody or antigen binding fragment binds to a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer) at least 20-fold greater than the MFI exhibited relative to binding to a BTN1a1 monomer (e.g., a glycosylated BTN1a1 monomer). In some embodiments, the antibody or antigen binding fragment binds to a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer) at least 25-fold greater than the MFI exhibited relative to binding to a BTN1a1 monomer (e.g., a glycosylated BTN1a1 monomer). In some embodiments, the antibody or antigen binding fragment binds to a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer) at least 30-fold greater than the MFI exhibited relative to binding to a BTN1a1 monomer (e.g., a glycosylated BTN1a1 monomer). In some embodiments, the antibody or antigen binding fragment binds to a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer) at least 40-fold greater than the MFI exhibited relative to binding to a BTN1a1 monomer (e.g., a glycosylated BTN1a1 monomer). In some embodiments, the antibody or antigen binding fragment binds to a BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer) at least 50-fold greater than the MFI exhibited relative to binding to a BTN1a1 monomer (e.g., a glycosylated BTN1a1 monomer).

In some embodiments, the antibody or antigen binding fragment preferentially binds glycosylated dimeric BTN1a1 relative to glycosylated monomeric BTN1a 1. Glycosylated BTN1a1 the two BTN1a1 monomers in the dimer may be independently glycosylated at the same position or at different positions. In some embodiments, one monomer in BTN1a1 dimer is not glycosylated. The glycosylated BTN1a1 monomer in the glycosylated BTN1a1 dimer may be glycosylated at positions N55, N215, and/or N449. In some embodiments, the glycosylated BTN1a1 monomer is glycosylated at position N55. In some embodiments, the glycosylated BTN1a1 monomer is glycosylated at position N215. In some embodiments, the glycosylated BTN1a1 monomer is glycosylated at position N449. In some embodiments, the glycosylated BTN1a1 monomer is glycosylated at positions N55 and N215. In some embodiments, the glycosylated BTN1a1 monomer is glycosylated at positions N55 and N449. In some embodiments, the glycosylated BTN1a1 monomer is glycosylated at positions N215 and N449. In some embodiments, the glycosylated BTN1a1 monomer is glycosylated at positions N55N215 and N449.

5.2.1 antibodies and other molecules with antigen-binding fragments

In some embodiments, an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody can be an IgG, IgM, IgA, IgD, or IgE. The anti-BTN 1A1 antibody or anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA) antibody can also be a chimeric antibody, an affinity matured antibody, a humanized antibody or a human antibody. The anti-BTN 1A1 antibody or anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA) antibody can also be a camelid antibody, an intrabody, an anti-idiotype (anti-Id) antibody. In some embodiments, an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody can be a polyclonal antibody or a monoclonal antibody.

Antibodies can be produced from any animal source, including avian and mammalian. In some embodiments, the antibody is a goat, mouse (e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse, or chicken. In addition, newer technologies allow the development and screening of human antibodies from human combinatorial antibody libraries. For example, phage antibody expression technology allows for the production of specific antibodies in the absence of animal immunization, as described in U.S. Pat. No.6,946,546, which is incorporated herein by reference in its entirety. These techniques are described in Marks (1992); stemmer (1994); gram et al (1992); barbas et al (1994) and Schier et al (1996) are further described, which are incorporated herein by reference in their entirety.

Methods for producing polyclonal antibodies in various animal species, as well as methods for producing various types of monoclonal antibodies, including humanization, chimerization and complete humanization, are well known in the art. For example, the following U.S. Pat. Nos. 3,817,837; 3,850,752, respectively; 3,939,350, respectively; 3,996,345; 4,196,265; 4,275, 149; 4,277,437; 4,366,241; 4,469,797, respectively; 4,472,509; 4,606,855, respectively; 4,703,003, respectively; 4,742,159, respectively; 4,767,720, respectively; 4,816,567; 4,867,973, respectively; 4,938,948, respectively; 4,946,778; 5,021,236, respectively; 5,164,296, respectively; 5,196,066, respectively; 5,223,409; 5,403,484; 5,420,253, respectively; 5,565,332; 5,571,698; 5,627,052; 5,656,434, respectively; 5,770,376, respectively; 5,789,208; 5,821,337; 5,844,091, respectively; 5,858,657, respectively; 5,861,155, respectively; 5,871,907, respectively; 5,969, 108; 6,054,297; 6,165,464, respectively; 6,365, 157; 6,406,867, respectively; 6,709,659, respectively; 6,709,873, respectively; 6,753,407, respectively; 6,814,965, respectively; 6,849,259, respectively; 6,861,572, respectively; 6,875,434, respectively; 6,891,024, respectively; 7,407,659, respectively; and 8,178,098, which are incorporated herein by reference in their entirety.

Molecules having antigen-binding fragments that immunospecifically bind to BTN1a1 or specifically bind to a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), including anti-BTN 1a1 antibody or anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody, may also be produced by any method known in the art for producing polypeptides, e.g., in vitro synthesis, recombinant DNA production, and the like. Humanized antibodies can be produced by recombinant DNA techniques. The antibodies described herein can also be produced using recombinant immunoglobulin expression techniques. Recombinant production of immunoglobulin molecules, including humanized antibodies, is described in U.S. Pat. No.4,816,397(Boss et al), U.S. Pat. Nos.6,331,415 and 4,816,567 (both to Cabilly et al), UK patent GB2,188,638(Winter et al) and UK patent GB2,209,757, which are incorporated herein by reference in their entirety. Techniques for recombinant expression of immunoglobulins, including humanized immunoglobulins, can also be found in Goeddel et al, Gene expression technology Methods in Enzymology Vol.185, academic Press (1991), and BorreBack, Antibody Engineering, W.H.Freeman (1992); which is incorporated herein by reference in its entirety. For additional information on the generation, design and expression of recombinant Antibodies, see Mayforth, design Antibodies, Academic Press, SanDiego (1993).

In certain embodiments, an anti-BTN 1A1 antibody or an anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA) antibody is a human antibody. Human antibodies can be made by a variety of methods known in the art, including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences (see U.S. Pat. Nos.4,444,887 and 4,716,111; and International publication Nos. WO98/46645, WO98/50433, WO98/24893, WO98/16654, WO96/34096, WO96/33735, and WO 91/10741). Human antibodies can be produced using transgenic mice that do not express functional endogenous immunoglobulins but express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells by random or by homologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable, constant and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy and light chain immunoglobulin genes can be rendered nonfunctional by homologous recombination by introducing the human immunoglobulin loci separately or simultaneously. In particular, homozygous deletion of the JH region prevents the production of endogenous antibodies. The modified embryonic stem cells were expanded and microinjected into blastocysts to generate chimeric mice. Chimeric mice are then bred to produce homozygous progeny expressing human antibodies. Transgenic mice are immunized with an antigen of choice, e.g., all or a portion of a BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA polypeptide, or a glycosylated BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA polypeptide, or a BTN1A1, GAL-1, GAL-9, NRP-2, or BTLA polypeptide dimer, using conventional methods. Monoclonal antibodies directed against the antigen can be obtained from immunized transgenic mice using conventional hybridoma technology (see, e.g., U.S. patent No.5,916,771). The human immunoglobulin transgene carried by the transgenic mice rearranges during B cell differentiation, followed by species switching and somatic mutation. Thus, using such techniques, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced. For a summary of such techniques for producing human antibodies, see Lonberg and Huszar (1995, int. Rev. Immunol.13:65-93, which is incorporated herein by reference in its entirety). For a detailed discussion of the techniques for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., international publications WO98/24893, WO96/34096 and WO 96/33735; and U.S. Pat. nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318 and 5,939,598, which are incorporated herein by reference in their entirety. Additionally, companies such as Abgenix, Inc (Freemont, Calif) and medrex (Princeton, n.j.) may use similar techniques as described above to provide human antibodies to selected antigens.

In some embodiments, an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody is a chimeric antibody, e.g., an antibody having antigen binding sequences from a non-human donor grafted onto heterologous non-human, or humanized sequences (e.g., framework and/or constant domain sequences). In one embodiment, the non-human donor is a rat. In one embodiment, the antigen binding sequence is synthetic, such as by mutagenesis (e.g., phage display screening of a human phage library, etc.). In one embodiment, the chimeric antibody may have a murine V region and a human C region. In one embodiment, the murine light chain V region is fused to a human kappa light chain. In one embodiment, the murine heavy chain V region is fused to a human IgG 1C region.

Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science229: 1202; oi et al, 1986, Biotechniques4: 214; gillies et al, 1989, J.Immunol.Methods125: 191-202; and U.S. Pat. nos.6,311,415, 5,807,715, 4,816,567 and 4,816,397; all of these patents are incorporated herein by reference in their entirety. Chimeric antibodies comprising one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art, including, for example, CDR grafting (EP 239400; International publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101 and 5,585,089), fitting or resurfacing (EP592,106; EP519,596; Padlan, 1991, Molecular Immunology28(4/5): 489-498; studnika et al, 1994, Protein Engineering7: 805; and Roguska et al, 1994, Proc. Natl.Acad.Sci.USA91:969) and chain shuffling groups (U.S. Pat. No.5,565,332). All of these patents are incorporated herein by reference in their entirety.

An exemplary procedure for producing recombinant chimeric anti-BTN 1a1 or anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibodies can include a) constructing an expression vector encoding and expressing an antibody heavy chain by conventional molecular biology methods, wherein the CDRs and variable regions of a murine anti-BTN 1a1 or anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) monoclonal antibody are fused to an Fc region derived from a human immunoglobulin, thereby producing a vector for expressing a chimeric antibody heavy chain; b) constructing an expression vector encoding and expressing an antibody light chain of a murine anti-BTN 1a1 or anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA) monoclonal antibody by conventional molecular biological methods, thereby producing a vector for expressing a chimeric antibody light chain; c) transferring the expression vector to a host cell by a conventional molecular biological method to produce a transfected host cell for expression of the chimeric antibody; and d) culturing the transfected cells by conventional cell culture techniques to produce the chimeric antibody.

An exemplary procedure for producing a recombinant humanized anti-BTN 1a1 antibody may comprise the steps of a) constructing an expression vector encoding and expressing an antibody heavy chain by conventional molecular biology methods, wherein a minimal portion of the CDR and variable region frameworks required to retain donor antibody binding specificity are derived from a non-human immunoglobulin, such as a murine anti-BTN 1a1 or anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA) monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for expressing a humanized antibody heavy chain; b) constructing an expression vector encoding and expressing an antibody light chain by conventional molecular biology methods, wherein a minimal portion of the CDR and variable region frameworks required to retain donor antibody binding specificity are derived from a non-human immunoglobulin, such as a murine anti-BTN 1a1 or anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA) monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector expressing a humanized antibody light chain; c) transferring the expression vector to a host cell by a conventional molecular biological method to produce a transfected host cell for expressing the humanized antibody; and d) culturing the transfected cells by conventional cell culture techniques to produce the humanized antibody.

For either exemplary method, the host cell may be co-transfected with an expression vector which may comprise different selectable markers, preferably identical except for the heavy and light chain coding sequences. The method provides for equal expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding both heavy and light chain polypeptides may be used. The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA or both. The host cell for expression of the recombinant antibody may be a bacterial cell such as E.coli, or more preferably a eukaryotic cell such as a Chinese Hamster Ovary (CHO) cell or a HEK-293 cell. The choice of expression vector depends on the choice of host cell and may be selected so that it has the desired expression and regulatory properties in the selected host cell. Other cell lines that may be used include, but are not limited to, CHO-K1, NSO and PER. C6(Crucell, Leiden, Netherlands). Furthermore, codon usage can be optimized when species-specific codon usage bias is required for the selected host cell and protein expression is enhanced. For example, for CHO cell expression, DNA encoding the antibody may incorporate codons preferentially used by Cricetulus griseus (from which chinese hamster ovary cells are derived). Codon-optimized methods can be used to promote increased expression in a desired host cell (see, e.g., Wohlgemuth, I. et al, Philos. Trans. R. Soc. Lond. BBiol. Sci.366(1580):2979-2986 (2011); Jestin, J.L. et al, J.mol. Evol.69(5):452-457 (2009); Bollenbach, T. et al, genome Res.17(4):401-404 (2007); Kurland, C.G. et al, prog nucleic Acid Res. mol. biol.31:191-219 (1984); Grosjean, H. et al, Gene18(3):199-209 (1982)).

In some embodiments, an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody can be a monoclonal antibody. In some embodiments, an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody can be a polyclonal antibody. The animal may be inoculated with an antigen, such as a BTN1A1 polypeptide or a glycosylated BTN1A1 polypeptide, a BTN1A1 dimer polypeptide, a GAL-1 polypeptide, a GAL-9 polypeptide, an NRP-2 polypeptide, or a BTLA polypeptide, to produce antibodies specific for the BTN1A1 polypeptide or the glycosylated BTN1A1 polypeptide, a BTN1A1 dimer, a GAL-1 polypeptide, a GAL-9 polypeptide, an NRP-2 polypeptide, or a BTLA polypeptide. Typically an antigen is bound or conjugated to another molecule to enhance the immune response. The conjugate can be any peptide, polypeptide, protein, or non-protein substance that binds to an antigen used to elicit an immune response in an animal. Antibodies produced in animals in response to antigen vaccination include a variety of different molecules produced by a variety of individual antibody-producing B lymphocytes (polyclonal antibodies). Given the correct conditions for polyclonal antibody production in an animal, most antibodies in the animal's serum recognize a collective epitope on the antigenic compound to which the animal has been immunized.

This specificity can be further enhanced by affinity purification, selecting only those antibodies that recognize the antigen or epitope of interest. The process of producing monoclonal antibodies (MAbs) can begin along the same route as polyclonal antibodies are made. In certain embodiments, rodents such as mice and rats are used to produce monoclonal antibodies. In certain embodiments, rabbit, sheep, or frog cells are used to produce monoclonal antibodies. The use of rats is well known and may provide certain advantages. Mice (e.g., BALB/c mice) are routinely used, generally resulting in a higher percentage of stable fusions.

Hybridoma technology involves the fusion of a single B lymphocyte from a mouse previously immunized with a BTN1a1 polypeptide, a glycosylated BTN1a1 polypeptide, a BTN1a1 dimer polypeptide, a GAL-1 polypeptide, a GAL-9 polypeptide, an NRP-2 polypeptide, or a BTLA polypeptide with an immortalized myeloma cell, typically a mouse myeloma. The technology provides a method for propagating single antibody producing cells for unlimited passage, so that an unlimited number of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity can be produced.

In one embodiment, the antibody is an immunoglobulin single variable domain derived from a camelid antibody, preferably from the heavy chain of a camelid antibody lacking a light chain, designated V_HH domain sequences or Nanobodies^TM。Nanobodies^TM(Nb) is the most abundant naturally occurring single chain antibodySmall functional fragments or Single variable domains (V)_HH) And are known to those skilled in the art. They are derived from the heavy chain-only antibodies seen in camels (Hamers-Casterman et al, Nature, 363(6428):446-8 (1993); Desmyter et al, Nat Struct Biol, 3(9):803-11 (1996)). In the "camel" family, immunoglobulins without light polypeptide chains are found. "Camel" includes camels of the old world (Bactrianus and dromedarius) and New world (e.g., alpaca (Lamapaccos), llama (Lama glama), alpaca (Lama guanicoe) and alpaca (Lama vicugna)). Single chain variable domain heavy chain antibody is defined as Nanobody^TMOr V_HH antibody. The small size and unique biophysical properties of Nbs are superior to conventional antibody fragments in that they are used to recognize unusual or cryptic epitopes and bind to cavities or active sites of protein targets. In addition, Nbs can be designed as multispecific and multivalent antibodies, linked to reporter molecules, or humanized. Nbs are stable, survive the gastrointestinal system, and can be easily manufactured.

Integrating two antigen-binding sites of different specificity into a single construct, bispecific antibodies have the ability to bind two discrete antigens of fine specificity together, and thus have great potential as therapeutic agents. Bispecific antibodies can be prepared by fusing two hybridomas, each capable of producing a different immunoglobulin. Bispecific antibodies can also be generated by linking two scFv antibody fragments while omitting the Fc portion present in a whole immunoglobulin. Each scFv unit in these constructs may consist of one variable domain from each of the heavy (VH) and light (VL) antibody chains, interconnected by a synthetic polypeptide linker, which is typically genetically engineered to be minimally immunogenic, while maintaining maximum resistance to proteolysis. Individual scFv units can be linked by a variety of techniques, including the incorporation of a short polypeptide (typically less than 10 amino acids) spacer bridging two scFv units, thereby generating a bispecific single chain antibody. Thus, the resulting bispecific single chain antibody is one which comprises two pairs of VH/VL pairs of different specificity on a single polypeptide chain, whereby the VH and VL domains in the respective scFv units are separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains, and the scFv units formed thereby are adjoined to each other by a polypeptide spacer which is kept short enough to prevent undesired association between, for example, the VH domain of one scFv unit and the VL of another scFv unit.

Examples of molecules having antigen binding fragments that immunospecifically bind to BTN1A1, glycosylated BTN1A1, BTN1A1 dimer, GAL-1, GAL-9, NRP-2, or BTLA include, but are not limited to, (i) Fab fragments consisting of VL, VH, CL, and CH1 domains; (ii) an "Fd" fragment consisting of the VH and CH1 domains; (iii) an "Fv" fragment consisting of the VL and VH domains of a single antibody; (iv) a "dAb" fragment consisting of a VH domain; (v) an isolated CDR region; (vi) f (ab')2 fragments, bivalent fragments including two linked Fab fragments; (vii) a single chain Fv molecule ("scFv"), wherein the VH domain and the VL domain are connected by a peptide linker that allows the two domains to associate to form a binding domain; (viii) bispecific single chain Fv dimers (see U.S. Pat. No.5,091,513); and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (U.S. patent application publication No. 20050214860), Fv, scFv or diabody molecules can be stabilized by binding a disulfide bond that links VH and VL domains. Mini antibodies with scFv linked to the CH3 domain can also be prepared (Hu et al, Cancer Res., 56(13):3055-61 (1996)).

Antibody-like binding peptidomimetics are also included in embodiments. Murali et al, Cell mol. biol., 49(2):209-216(2003) describe "antibody-like binding peptidomimetics" (ABiPs) which are peptides that are used as attenuated antibodies and which have certain advantages of long serum half-lives and less cumbersome synthetic methods, which are incorporated herein by reference in their entirety.

In certain embodiments, the derivatives comprise fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule.

In some embodiments, the molecules provided herein can be chemically modified, for example, by covalently linking any type of molecule to an antibody. For example, but not limited to, antibody derivatives include chemically modified antibodies, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be made by known techniques, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. In addition, the antibody may contain one or more non-canonical amino acids.

The molecules provided herein can have framework regions (e.g., human or non-human fragments) known to those of skill in the art. The framework regions may be, for example, naturally occurring or shared framework regions. In particular embodiments, the framework regions of the antibodies provided herein are human (see, e.g., Chothia et al, 1998, J.mol.biol.278: 457-. See also Kabat et al (1991)Sequences of Proteins of Immunological Interest(U.S. department of Health and Human Services, Washington, d.c.) 5 th edition.

In another aspect, provided herein are molecules having an antigen-binding fragment that competitively blocks (e.g., in a dose-dependent manner) the BTN1a1 epitope of an anti-BTN 1a1 antibody, the GAL-1 epitope of an anti-GAL 1 antibody, the anti-GAL 9 epitope of an anti-GAL 9 antibody, the anti-NRP-2 epitope of an anti-NRP 2 antibody, or the BTLA epitope of an anti-BTLA antibody. The molecule may be an antibody. The antibody may be a monoclonal antibody. The antibody may be a humanized antibody.

In some embodiments, provided herein are anti-BTLA antibodies that competitively block (e.g., in a dose-dependent manner) a BTN1a1 epitope described herein, an anti-GAL-1 epitope of an anti-GAL-1 antibody, an anti-GAL-9 epitope of an anti-GAL-9 antibody, an anti-NRP-2 epitope of an anti-NRP-2 antibody, or an anti-BTLA epitope of an anti-BTLA antibody.

In certain embodiments, the molecules provided herein have high affinity for BTN1a1, glycosylated BTN1a1, BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer), GAL-1, GAL-9, NRP-2, BTLA, or a polypeptide or polypeptide fragment or epitope thereof. In one embodiment, the molecule provided herein can be an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody with higher affinity for BTN1a1 or the BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) than known antibodies (e.g., commercially available monoclonal antibodies discussed elsewhere herein). In particular embodiments, the molecules provided herein can be anti-BTN 1a1 antibodies or anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibodies with 2-10 fold (or greater) greater affinity for BTN1a1, GAL-1, GAL-9, NRP-2, or BTLA antigens than known anti-BTN 1a1 antibodies or known anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibodies, as determined by the techniques described herein or known to those of skill in the art (e.g., Biacore assay). According to these embodiments, in one embodiment, the affinity of the antibody is assessed by Biacore assay.

In certain embodiments, the molecules provided herein can have an antigen-binding fragment that binds to BTN1a1, glycosylated BTN1a1, BTN1a1 dimer (e.g., a glycosylated BTN1a1 dimer), GAL-1, GAL-9, NRP-2, BTLA, or a polypeptide or polypeptide fragment or epitope, with a dissociation constant (K) thereof_D) Not greater than 1 μ M, not greater than 100nM, not greater than 10nM, not greater than 1nM, or not greater than 0.1 nM. In some embodiments, the molecule provided herein can be K_DAn anti-BTN 1A1 antibody or an anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody of no greater than 500 nM. In some embodiments, the molecule provided herein can be K_DAn anti-BTN 1A1 antibody or an anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody of no greater than 200 nM. In some embodiments, the molecule provided herein can be K_DAn anti-BTN 1A1 antibody or an anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, RP-2, or BTLA) antibody of no greater than 100 nM. In some embodiments, the molecule provided herein can be K_DAn anti-BTN 1A1 antibody or an anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody of no greater than 50 nM. In some embodiments, the molecule provided herein can be K_DAn anti-BTN 1A1 antibody or an anti-BTN 1A1 ligand (e.g., GAL-1, Gal-9, NRP-2, or BTLA) antibody of no greater than 20 nM. In some embodiments, the molecule provided herein can be K_DAn anti-BTN 1A1 antibody or an anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody of no greater than 10 nM. In some embodiments, the molecule provided herein can be K_DAn anti-BTN 1A1 antibody or an anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody of no greater than 5 nM. In some embodiments, the molecule provided herein can be K_DAn anti-BTN 1A1 antibody or an anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody of no greater than 2 nM. In some embodiments, the molecule provided herein can be K_DAn anti-BTN 1A1 antibody or an anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody of no greater than 1 nM. In some embodiments, the molecule provided herein can be K_DAn anti-BTN 1A1 antibody or an anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody at no greater than 0.5 nM. In some embodiments, the molecule provided herein can be K_DAn anti-BTN 1A1 antibody or an anti-BTN 1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody at no greater than 0.1 nM.

In certain embodiments, the molecules provided herein can block or neutralize the activity of a BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA). The molecule may be a neutralizing antibody. The neutralizing antibodies block binding of BTN1A1 to a natural ligand such as GAL-1, GAL-9, NRP-2 or BTLA and inhibit the signaling pathway and/or other physiological activity mediated by the BTN1A1 or BTN1A1-BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA) complex. In neutralization assays, the IC50 for neutralizing antibodies can be in the range of 0.01-10 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 10 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 8 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 6 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 4 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 2 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 1 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 0.8 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 0.6 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 0.4 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 0.2 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 0.1 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 0.08 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 0.06 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 0.04 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 0.02 μ g/ml. The IC50 of the neutralizing antibody may be no greater than 0.01 μ g/ml.

The molecules provided herein having an antigen-binding fragment that immunospecifically binds BTN1a1 (e.g., glycosylated BTN1a1 or BTN1a1 dimer) or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA) can be an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA) antibody. Antibodies provided herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, polypeptidesSpecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the foregoing. Non-limiting examples of functional fragments include single chain fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F (ab') fragments, F (ab)₂Fragment F (ab')₂Fragments, disulfide-linked Fv (SdFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies and minibodies.

In particular, the molecules provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., molecules that contain antigen binding fragments that immunospecifically bind to BTN1a1 (e.g., glycosylated BTN1a1 or BTN1a1 dimer) or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA). The immunoglobulin molecules provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or immunoglobulin molecule subclass.

The molecules provided herein can be monospecific, bispecific, trispecific antibodies or antibodies with higher multispecific. The multispecific antibody may be specific for a BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), or may be specific for both a BTN1a1 polypeptide or a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) polypeptide and a heterologous epitope (e.g., a heterologous polypeptide or a solid support material). In particular embodiments, the antibodies provided herein are monospecific for a given epitope of a BTN1a1 polypeptide or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) polypeptide and do not bind to other epitopes.

5.2.2 modifications and derivatives

Antigen-binding fragments with immunospecific binding to BTN1a1 (e.g., glycosylated BTN1a1 or BTN1a1 dimer) or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA) can be further improved by screening for variants exhibiting desired properties. For example, such improvements can be accomplished using various phage display methods known in the art. In the phage display method, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding them. In a particular embodiment, such phage may be used to display antigen binding fragments, such as Fab and Fv or disulfide-stabilized Fv, expressed from whole libraries or combinatorial antibody libraries (e.g., human or murine). Phage expressing an antigen-binding fragment that binds an antigen of interest can be selected or identified with the antigen, for example, using a labeled antigen or antigen bound or captured to a solid surface or bead. The phage used in these methods are typically filamentous phage, including fd and ML 3. The antigen binding fragment is expressed as a protein recombinantly fused to the phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare antibodies or other molecules having the antigen-binding fragments described herein include Brinkman et al, J immunol methods, 182:41-50 (1995); ames et al, J.Immunol.methods, 184:177-186 (1995); kettleborough et al, Eur.J. Immunol, 24: 952-; persic et al, Gene, 187:9-18 (1997); burton et al, adv. Immunol.57:191-280 (1994); PCT publication WO 92/001047; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753, respectively; 5,821,047, respectively; 5,571,698; 5,427,908; 5,516,637; 5,780,225, respectively; 5,658,727, respectively; 5733743 and 5969108; all of which are incorporated herein by reference in their entirety.

As described in the above references, following phage selection, antibody coding regions from the phage can be isolated and used to produce whole antibodies, including humanized antibodies or any other desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail below. For example, recombinant production of Fab, Fab 'and F (ab')₂The techniques of (a) can also be used using methods known in the art, for example, PCT publication WO 92/22324; mullinax, R.L., et al, BioTechniques, 12(6):864-869 (1992); and Sawai et al, AM.J.reprod.Immunol.34:26-34 (1995); better, M et al, Science240: 1041-; the entire contents of all of these documents are hereby incorporated by referenceIncorporated by reference. Examples of techniques that can be used to produce single chain Fv's and antibodies include U.S. Pat. nos.4,946,778 and 5,258,498; huston, J.S. et al, Methods in Enzymology203:46-88 (1991); shu, L, et al, Proc. Natl. Acad. Sci. (USA)90: 7995-; and those described in Skerra.A. et al, Science240: 1038-; all of which are incorporated herein by reference in their entirety.

Phage display technology can be used to increase the affinity of anti-BTN 1a1 antibodies or BTN1a1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibodies or other molecules with antigen binding fragments that immunospecifically bind to BTN1a1 (e.g., glycosylated BTN1a1 or BTN1a1 dimer) or BTN1a1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) as described herein. The techniques can be used to obtain high affinity antibodies that can be used in the combinatorial methods described herein. The technique, known as affinity maturation, uses mutagenesis or CDR walking and uses re-selection of such receptors or ligands (or their extracellular domains) or antigenic fragments thereof to identify antibodies that bind with higher affinity to the antigen than the original or parent antibody (see, e.g., Glaser, S.M. et al, J.Immunol.149: 3903-. Mutagenesis of the entire codon rather than a single nucleotide results in a semi-random repertoire of amino acid mutations. Libraries consisting of pools of variant clones, each differing by a single amino acid change in a single CDR, can be constructed containing variants representing each possible amino acid substitution for each CDR residue. Mutants with improved binding affinity for antigen can be screened by contacting the immobilized mutants with a labeled antigen. Any screening method known in the art can be used to identify mutant antibodies (e.g., ELISA) with increased affinity for the antigen (see, e.g., Wu, H. et al, Proc. Natl. Acad. Sci. (USA)95(11): 6037-.

Random mutagenesis can be used in conjunction with phage display methods to identify improved CDRs and/or variable regions. Phage display technology can also be used to increase (or decrease) CDR affinity by directed mutagenesis (e.g., affinity maturation or "CDR-walking"). The technique uses a target antigen or antigenic fragment thereof to identify antibodies with CDRs that bind antigen with higher (or lower) affinity when compared to the original or parent antibody (see, e.g., Glaser, S.M. et al, J.Immunol.149: 3903-.

Methods for achieving such affinity maturation are described, for example, in Krause, J.C., et al, MBio.2(1) Pii: e00345-10.doi:10.1128/mBio.00345-10 (2011); kuan, C.T., et al, int.J.cancer 10.1002/ijc.25645; hackel, B.J., et al, J.mol.biol.401(1):84-96 (2010); montgomery, D.L., et al, MAbs1(5): 462-; gustchia, E.et al, Virology 393(1): 112-; finlay, W.J., et al, J.mol.biol.388(3):541-558 (2009); bostrom, J.et al, MethodsMol.biol.525: 353-; steidl, S. et al, mol.Immunol.46(1): 135-' 144 (2008); and Barderas, R. et al, Proc. Natl. Acad. Sci. (USA)105(26): 9029-; all of which are incorporated herein by reference in their entirety.

Also provided herein are derivatives of any of the above molecules having an antigen binding fragment that immunospecifically binds BTN1a1 (e.g., glycosylated BTN1a1 or BTN1a1 dimer) or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), which may be an anti-BTN 1a1 antibody or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody, but has one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications relative to the "parent" (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. Such amino acids may be glycosylated (e.g., altered mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-ethanoneuraminic acid, etc.), acetylated, PEGylated, phosphorylated, amidated, derivatized with known protecting/blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, etc. In some embodiments, the altered carbohydrate modification modulates one or more of solubilization of the antibody, promotion of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In some embodiments, the altered carbohydrate modification enhances antibody-mediated effector function relative to an antibody lacking the carbohydrate modification. Carbohydrate modifications which result in antibody-mediated alteration of effector function are well known in the art (see, for example, shelves, R.L. et al, J.biol. chem.277(30): 26733. 26740 (2002); Davies J et al, Biotechnology & Bioengineering, 74(4): 288-.

In some embodiments, the humanized antibody is a derivative antibody. Such humanized antibodies comprise amino acid residue substitutions, deletions or additions in one or more non-human CDRs. The humanized antibody derivative may have substantially the same binding, better binding, or poorer binding than the non-derivative humanized antibody. In some embodiments, one, two, three, four or five amino acid residues of a CDR are mutated, e.g., substituted, deleted or added.

The molecules and antibodies described herein can be modified by chemical modifications using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. In one embodiment, the derivative molecule or derivative antibody has a similar or identical function as the parent molecule or antibody. In another embodiment, the derivative molecule or derivative antibody exhibits altered activity relative to the parent molecule or parent antibody. For example, the derivative antibody (or fragment thereof) may bind its epitope more tightly or be more resistant to proteolysis than the parent antibody.

Substitutions, additions or deletions in the derivatized antibody may be in the Fc region of the antibody and thus may be used to alter the binding affinity of the antibody to one or more fcyrs. Methods of modifying antibodies that bind to one or more Fc γ rs are known in the art, see, e.g., PCT publications WO04/029207, WO04/029092, WO04/028564, WO99/58572, WO99/51642, WO98/23289, WO89/07142, WO88/07089 and U.S. patent nos. 5,843,597 and 5,642,821; all of these patents are incorporated herein by reference in their entirety. In some embodiments, the antibody or other molecule has an altered affinity for an activated Fc γ R, e.g., Fc γ RIIIA. Preferably, such modifications also have altered Fc-mediated effector function. Modifications that affect Fc-mediated effector function are well known in the art (see U.S. Pat. No.6,194,551 and WO 00/42072). In some embodiments, the modification of the Fc region results in an antibody having altered antibody-mediated effector function, altered binding to other Fc receptors (e.g., Fc activation receptors), altered antibody-dependent cell-mediated cytotoxicity (ADCC) activity, altered C1q binding activity, altered complement-dependent cytotoxicity (CDC) activity, altered phagocytosis activity, or any combination thereof.

ADCC is a cell-mediated reaction in which antigen-nonspecific cytotoxic cells that express FcR (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize antibodies that bind to the surface of target cells, subsequently leading to lysis (i.e., "killing") of the target cells. The initial mediating cells are NK cells. NK cells express only Fc γ RIII, where Fc γ RIIIA is the activating receptor and Fc γ RIIIB is the inhibiting receptor; monocytes express Fc γ RI, Fc γ RII and Fc γ RIII (ravatch et al (1991) Annu. Rev. Immunol., 9: 457-92). ADCC activity can be expressed as the concentration of antibody or Fc fusion protein at which lysis of the target cells is half maximal. Thus, in some embodiments, the concentration of the antibody or Fc fusion protein of the invention wherein the level of lysis is the same as the half-maximal level of lysis of the wild-type control is at least 2,3, 5, 10, 20, 50, 100 fold lower than the concentration of the wild-type control itself. In addition, in some embodiments, an antibody or Fc fusion protein of the invention can exhibit higher maximum target cell lysis compared to a wild-type control. For example, the antibody or Fc fusion protein may have a maximum target cell lysis 10%, 15%, 20%, 25% or more greater than the wild-type control.

The molecules and antibodies described herein can be modified to have enhanced potency. In some embodiments, the molecule and antibody effector functions are modified, e.g., to enhance ADCC and/or Complement Dependent Cytotoxicity (CDC). In some embodiments, these therapeutic molecules or antibodies have enhanced interactions with killer cells bearing Fc receptors. Enhancement of effector function, such as ADCC, can be achieved by various methods, including the introduction of one or more amino acid substitutions in the Fc region. In addition, cysteine residues may be introduced into the Fc region, allowing interchain disulfide bonds to form in the region. The homodimeric antibody may also have improved internalization capability and/or increased CDC and ADCC. Caron et al, J.Exp Med., 176:1191-95(1992) and shop, B.J.Immunol., 148:2918-22 (1992). A hetero-bifunctional cross-linker may also be used to prepare the homodimeric antibody Wolff et al, Cancer Research, 53:2560-65(1993), with enhanced anti-Cancer activity. In addition, antibodies or molecules with dual Fc regions can be engineered to have enhanced CDC and ADCC capabilities. Stevenson et al, Anti-cancer degdesign 3:219-30 (1989).

The glycosylation pattern of the Fc region can also be engineered. Many forms of antibody glycosylation are reported to have a positive impact on effector functions, including ADCC. Thus, engineering the carbohydrate component of the Fc region, particularly to reduce core fucosylation, may also have enhanced therapeutic efficacy. Shinkawa t. et al, J biol. chem., 278:3466-73 (2003); niwa R, et al, cancer Res.64:2127-33 (2004); okazaki A et al, Jmol. chem.336:1239^19 (2004); and Shields RL et al, Jbiol. chem.277:26733-40 (2002). The antibodies or molecules having a selected glycoform described herein can be produced by a variety of means, including the use of glycosylation pathway inhibitors, mutant cell lines that lack or reduce specific enzymatic activity in the glycosylation pathway, engineered cells with enhanced or knocked-out gene expression in the glycosylation pathway, and in vitro engineering with glycosidases and glycosyltransferases. Methods of modifying the glycosylation of the Fc region and enhancing the therapeutic efficacy of antibodies or other molecules having antigen-binding fragments are known in the art. Rothman et al, molecular immunology 26:1113-1123 (1989); m Mana et al, Nature Biotechnology 17:176-180 (1999); shields et al, JBC277:26733-26740 (2002); shinkawa et al, JBC278: 3466-; bischoff et al, J.biol.chem.265(26):15599-15605 (1990); U.S. Pat. Nos.6,861,242 and 7,138,262 and U.S. publication No. 2002/0124652; all of which are incorporated herein by reference in their entirety. It will be appreciated by those of ordinary skill in the art that the antibodies and molecules provided herein can be modified by any method known in the art to have enhanced therapeutic efficacy.

Derivative molecules or antibodies may also have an altered half-life (e.g., serum half-life) of the parent molecule or antibody in a mammal, preferably a human. In some embodiments, such a change results in a half-life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-life of a humanized antibody or other molecule in a mammal, preferably a human, results in a higher serum titer of the antibody or other molecule in the mammal, thus reducing the frequency of administration of the antibody or other molecule and/or reducing the concentration of the antibody or other molecule to be administered. Molecules or antibodies with increased in vivo half-life can be produced by techniques known to those skilled in the art. For example, molecules or antibodies with increased in vivo half-life may be generated by amino acid residue modifications (e.g., substitutions, deletions or additions) identified as being involved in the interaction between the Fc domain and the FcRn receptor. The humanized antibodies described herein can be engineered to increase biological half-life (see, e.g., U.S. patent No.6,277,375). For example, the humanized antibodies described herein may be engineered in the Fc-hinge domain to have increased in vivo or serum half-life.

By attaching a polymer molecule, such as high molecular weight polyethylene glycol (PEG), to the antibody or antibody fragment, the molecules or antibodies with increased in vivo half-life described herein can be produced. PEG can be attached to the molecule or antibody by site-specific coupling of PEG to the N-or C-terminus of the molecule or antibody or by epsilon-amino groups present on lysine residues, with or without the use of multifunctional linkers. Linear or branched polymer derivatizations with minimal loss of biological activity may be used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of the PEG molecule to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by, for example, size exclusion or ion exchange chromatography.

The molecules or antibodies described herein can also be modified by the methods and coupling agents described by Davis et al (see U.S. patent 4,179,337) to provide compositions that can be injected into the circulatory system of a mammal without a substantial immunogenic response. Removal of the Fc portion can reduce the likelihood that the antibody fragment will elicit an undesirable immune response, and thus, Fc-free antibodies can be used for prophylactic or therapeutic treatment. As noted above, antibodies can also be constructed as chimeric, partially or fully human to reduce or eliminate adverse immunological consequences resulting from administration to an animal of antibodies produced in or having sequences from other species.

5.2.3 fusion proteins and conjugates

Provided herein are molecules having antigen-binding fragments that immunospecifically bind to BTN1a1 (e.g., glycosylated BTN1a1 or BTN1a1 dimer) or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA), including anti-BTN 1a1 antibodies and anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA) antibodies. In some embodiments, such molecules are expressed as fusion proteins with other proteins, or chemically conjugated to another moiety.

In some embodiments, the molecule is a fusion protein having an Fc portion, which can vary by isotype or subclass, can be chimeric or hybrid, and/or can be modified, for example, to improve effector function, half-life control, tissue accessibility, enhance biophysical properties such as stability, and increase production efficiency (and at a lower cost). Many modifications that can be used to construct the disclosed fusion proteins and methods for making them are known in the art, see, e.g., Mueller, J.P. et al, mol.Immun.34(6): 441-. In some embodiments, the Fc region is a native IgG1, IgG2, or IgG4Fc region. In some embodiments, the Fc region is a hybrid, e.g., a chimera with an IgG2/IgG 4Fc constant region. Modifications to the Fc region include, but are not limited to, IgG4 modified to prevent binding to Fc receptors and complement, IgG1 modified to improve binding to one or more Fc receptors, IgG1 modified to minimize effector function (amino acid changes), IgG1 with altered/no glycans (typically by altering the expression host), and IgG1 with altered pH-dependent binding to FcRn. The Fc region may include the entire hinge region, or less than the entire hinge region.

Another embodiment includes IgG2-4 hybrids and IgG4 mutants that have reduced binding to FcR, which increases their half-life. Representative IG2-4 hybrids and IgG4 mutants are described in Angal et al, mol. Immunol.30(1):105-108 (1993); muller et al, mol.Immun.34(6):441-452 (1997); and U.S. patent nos.6,982,323; all of which are incorporated herein by reference in their entirety. In some embodiments, the IgG1 and/or IgG2 domains are deleted, e.g., Angal et al describe IgG1 and IgG2 with proline substituted for serine at position 241.

In some embodiments, the molecule is a polypeptide having at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids.

In some embodiments, provided herein are molecules having antigen-binding fragments that immunospecifically bind to BTN1a1 (e.g., glycosylated BTN1a1 or BTN1a1 dimer) or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), linked or covalently bound to or forming a complex with at least one moiety. Such moieties may be, but are not limited to, moieties that increase the efficacy of the molecule as a diagnostic or therapeutic agent. In some embodiments, the moiety can be an imaging agent, a toxin, a therapeutic enzyme, an antibiotic, a radiolabeled nucleotide, or the like.

The molecules provided herein canIncluding therapeutic moieties (or one or more therapeutic moieties) the molecules provided herein may be antibodies which are conjugated to or recombinantly fused to therapeutic moieties, e.g., cytotoxins, e.g., cytostatic or cytocidal agents, therapeutic agents or radioactive metal ions, e.g., α -emitters cytotoxins or cytotoxic agents including any agent which is detrimental to cells the therapeutic moieties include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dactinomycin), alkylating agents (e.g., methyldichloroethylamine, thiotepa chlorambucil, milflange, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin C and cis-dichlorodiamidoplatinum (II) (DDP) and cisplatin), anthracyclines (e.g., daunorubicin and doxorubicin), actinomycin (e.g., actinomycin (formerly daunorubicin), actinomycin (formerly), phytostatin, phytoestrogen, phytoPuromycin, and analogs or homologs thereof, and compounds disclosed in U.S. Pat. Nos., 6,218,410Those disclosed in the patent nos.6,277,832, 5,998,596, 5,885,834, 5,734,033 and 5,618,709); adenosine deaminase inhibitors (e.g., fludarabine phosphate and 2-chlorodeoxyadenosine); ibritumomab tiuxetan

Tositumomab

) And pharmaceutically acceptable salts, solvates, clathrates and prodrugs thereof.

Further, the molecules provided herein may be antibodies conjugated or recombinantly fused to therapeutic or pharmaceutical moieties that modify a given biological response, such proteins may include, for example, toxins, such as abrin, ricin A, Pseudomonas exotoxin, cholera toxin, or diphtheria toxin, proteins, such as tumor necrosis factor, gamma-interferon, α -interferon, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic agents, such as, for example, TNF-gamma, AMI I (see, for example, International publication No. WO97/33899), AMI II (see, for example, International publication No. WO97/34911), FasHa ligands (see, for example, Jakashi et al.,1994, J.Immunol.,6: 7-la, and Interleukin-IL-5, such as, interleukin-IL-1, interleukin-5, interleukin-IL-2, interleukin-IL-5, interleukin-IL-G, and a-G-.

Furthermore, the antibodies provided herein may be conjugated to a therapeutic moiety such as a radioactive metal ion, such as an emitter, such as α -emitter,²¹³bi or a macrocyclic chelator for conjugating radiometal ions to polypeptides, including but not limited to,¹³¹In，¹³¹LU，¹³¹Y，¹³¹HO，¹³¹sm. In certain embodiments, the macrocyclic chelator is 1,4,7, 10-tetraazacyclododecane-N, N ', N ", N'" -tetraacetic acid (DOTA), which can be attached to an antibody via a linker molecule. Such linker molecules are well known in the art and described in Denadro et al, 1998, Clin Cancer Res.4(10): 2483-90; peterson et al, 1999, bioconjugate. chem.10(4): 553-7; and Zimmerman et al, 1999, Nucl. Med. biol.26(8):943-50, which are incorporated by reference in their entirety.

Therapeutic moieties or drugs conjugated or recombinantly fused to antibodies provided herein that immunospecifically bind to BTN1a1 (e.g., glycosylated BTN1a1 or BTN1a1 dimer) or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2 or BTLA) should be selected to achieve the desired prophylactic or therapeutic effect. In certain embodiments, the antibody is a modified antibody. The clinician or other medical personnel should consider factors such as the nature of the disease, the severity of the disease, and the condition of the subject in deciding which therapeutic moiety or drug to use for conjugation or recombinant fusion to the antibody provided herein.

In certain embodiments, the moiety can be an enzyme, a hormone, a cell surface receptor, a toxin (e.g., abrin (abrin), ricin a, pseudomonas exotoxin (i.e., PE-40), diphtheria toxin, ricin, gelonin (gelonin), or pokeweed antiviral protein), a protein (e.g., tumor necrosis factor, interferon(e.g., α interferon, β interferon), nerve growth factor, platelet-derived growth factor, tissue plasminogen activator or apoptosis agent (e.g., tumor necrosis factor- α, tumor necrosis factor- β)), biological response modifiers (e.g., lymphokines (e.g., interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6")), granulocyte macrophage colony-stimulating factor ("GM-CSF"), granulocyte colony-stimulating factor ("G-CSF") or macrophage colony-stimulating factor, ("M-CSF")), or growth factors (e.g., growth hormone ("GH")), cytotoxins (e.g., cytostatic or cytocidal agents, e.g., paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emidine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyactinodione, anthraquinone, aureoxypicrin, aureoxydine, monocrotaxin, antimine, antimitone, antimycosine, antimine, antimycosine,

(carmustine; BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cisplatin (II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., actinomycin (formerly namycin), bleomycin, mithramycin and Anthranthromycin (AMC), or antimitotic agents (e.g., vincristine and vinblastine).

The techniques for coupling such therapeutic moieties to antibodies are well known; see, For example, Amon et al, "Monoclonal Antibodies For immunotargeting of Drugs In Cancer Therapy", inMONOCLONAL ANTIBODIES AND CANCER THERAPY, Reisfeld et al (eds.),1985, pp.243-56, AlanR.Liss, Inc.); hellstrom et al, "Antibodies For Drug Delivery", in CONTROL EDDRUG DELIVERY (2nd Ed.), Robinson et al (eds.),1987, pp.623-53, Marcel Dekker, Inc.); thorpe, "ANTIBODIES Of cytotoxin Agents In Cancer Therapy: AReview", inMONOCLONAL ANTIBODIES'84: BIOLOGICAL AND CLINICAL APPLICATIONS, Pincher et al (eds.),1985, pp.475-506); "Analysis, Results, And d Future productive Of therapeutic Use Of radioactive Antibody In Cancer Therapy", In MONOCLONALANTIBIDIES FOR CANCER DETECTION AND THERAPY, Baldwin et al (eds.),1985, pp.303-16, Academic Press; thorpe et al, Immunol.Rev.62:119-158 (1982); carter et al, Cancer J.14(3): 154-; alley et al, curr, Opin, chem, biol.14(4): 529-; carter et al, am. Assic. cancer Res. Educ. book.2005(1): 147-; carter et al, cancer J.14(3):154-169 (2008); chari, Acc. chem Res.41(1):98-107 (2008); doronina et al, nat. Biotechnol.21(7):778-784 (2003); ducry et al, bioconjugugChem.21 (1):5-13 (2010); senter, curr, Opin, chem, biol.13(3):235-244 (2009); and Teicher, Curr Cancer drug targets.9(8): 982-.

In some embodiments, the molecules described herein can be conjugated to a marker, such as a peptide, to facilitate purification. In some embodiments, the marker is a hexa-histidine peptide, a hemagglutinin "HA" tag, which corresponds to an epitope derived from influenza hemagglutinin protein (Wilson, I.A. et al, Cell, 37:767-778(1984)), or a "FLAG" tag (Knappik, A et al, Biotechniques17(4):754-761 (1994)).

In some embodiments, the moiety may be an image reagent that can be detected in an assay. Such an imaging agent may be an enzyme, prosthetic group, radiolabel, nonradioactive paramagnetic metal ion, hapten, fluorescent tag, phosphorescent molecule, chemiluminescent molecule, chromophore, luminescent molecule, bioluminescent molecule, photoaffinity molecule, colored particle or ligand, such as biotin.

In some embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaliSex phosphatase, β -galactosidase or acetylcholinesterase, prosthetic group complexes including but not limited to streptavidin/biotin and avidin/biotin, fluorescent materials including but not limited to umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin, luminescent materials such as but not limited to luminol, bioluminescent materials including but not limited to luciferase, fluorescein and aequorin, radioactive materials including but not limited to bismuth (R), (B), (C), (²¹³Bi), carbon (C: (B)¹⁴c) Chromium (A), (B), (C), (⁵¹Cr, cobalt (b)⁵⁷CO), fluorine (¹⁸f) Gadolinium (a)¹⁵³Gd，¹⁵⁹Gd, gallium (b)⁶⁸Ga，⁶⁷Ga), germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium (¹¹⁵In，¹¹³In，¹¹²In，¹¹¹In), iodine (¹³¹I，¹²⁵I，¹²³I，¹²¹I) Lanthanum (a)¹⁴⁰La), lutetium (¹⁷⁷Lu), manganese (⁵⁴Mn, molybdenum (C)⁹⁹Mo), palladium (¹⁰³Pd), phosphorus (C)³²P), praseodymium (¹⁴²Pr), promethium (M)¹⁴⁹Pm), rhenium (¹⁸⁶Re，¹⁸⁸Re), rhodium (II)¹⁰⁵Rh), ruthenium (II)⁹⁷Ru), samarium (¹⁵³Sm, scandium (⁴⁷Sc), selenium (⁷⁵Se), strontium (⁸⁵Sr, sulfur (S) <³⁵S), technetium (C)⁹⁹Tc), thallium (²⁰¹Ti, tin (b)¹¹³Sn，¹¹⁷Sn), tritium (³h) Xenon (a)¹³³Xe), ytterbium (¹⁶⁹Yb，¹⁷⁵Yb), yttrium (b)⁹⁰Y), zinc (⁶⁵Zn); various positron emitting tomographic positron emitting metals and non-radioactive paramagnetic metal ions are used.

Some conjugation methods involve the use of metal chelate complexes that are linked to the antibody using, for example, organic chelators, such as diethylenetriaminepentaacetic anhydride (DTPA), ethylenetriaminetetraacetic acid, N-chloro-p-toluenesulfonamide, and/or tetrachloro-3-6 α -diphenylglycyl-3 monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate.

Such heteroconjugate antibodies can additionally bind to a hapten (e.g., fluorescein), or a cellular marker (e.g., 4-1-BB, B7-H4, CD4, CD8, CD14, CD25, CD27, CD40, CD68, CD163, CTLA4, GITR, LAG-3, OX40, TIM3, TIM4, TLR2, LIGHT, ICOS, B7-H3, B7-H7, B7-H7CR, CD70, CD47) or a cytokine (e.g., IL-7, IL-15, IL-12, IL-4TGF- β -10, IL-17, IFNY, Flt3, BLys 21).

The molecules described herein can be attached to a solid support, which can be used for immunoassay or purification of a target antigen or other molecule capable of binding to a target antigen immobilized on the support by binding to an antibody or antigen binding fragment described herein. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

Also provided herein are nucleic acid molecules (DNA or RNA) encoding any such antibodies, antigen-binding fragments, and molecules having an antigen-binding fragment that immunospecifically binds BTN1a1 (e.g., glycosylated BTN1a1 or BTN1a1 dimer). Also provided herein are vector molecules (e.g., plasmids) capable of delivering or replicating the nucleic acid molecules. The nucleic acid may be single-stranded, double-stranded, and may comprise both single-stranded and double-stranded portions.

Antibody-drug conjugates (ADCs)

Since the molecules provided herein can result in internalization of BTN1a1 into a cell, also provided herein are antibody-drug conjugates (ADCs) comprising any of the anti-BTN 1a1 antibodies described herein.

In some embodiments, provided herein are antibody-drug conjugates comprising antibody-drug conjugates of the following formulas (Ia) and (Ib):

or a pharmaceutically acceptable salt thereof;

wherein:

a is a molecule having an antigen-binding fragment;

the two depicted cysteine residues are from the open cysteine-cysteine disulfide bond in a;

each of X and X' is independently O, S, NH or NR¹Wherein R is¹Is C_1-6An alkyl group;

W_ais ═ N-, ═ CH-, ═ CHCH-₂-、＝C(R²) -, or ═ CHCH (R)²)-；W_b-NH-、-N(R¹)-、-CH₂-、-CH₂-NH-、-CH₂-N(R¹)-、-CH₂CH₂-、-CH(R²) -, or-CH₂CH(R²) -; wherein R is¹And R²Independently is C_1-6An alkyl group;

CTX is a cytotoxin;

r is any chemical group, or R is absent;

L¹、L²and L³Is independently a linker selected from the group consisting of: -O-, -C (O) -, -S (O)₂-、-NH-、-NCH₃-、-(CH₂)_q-、-NH(CH₂)₂NH-、-OC(O)-、-CO₂-、-NHCH₂CH₂C(O)-、-C(O)NHCH₂CH₂NH-、-NHCH₂C(O)-、-NHC(O)-、-C(O)NH-、-NCH₃C(O)-、-C(O)NCH₃-、-(CH₂CH₂O)_p、-(CH₂CH₂O)_pCH₂CH₂-、-CH₂CH₂-(CH₂CH₂O)_p-、-OCH(CH₂O-)₂、-(AA)_r-, cyclopentyl, cyclohexyl, unsubstituted phenylene, and phenylene substituted with 1 or 2 substituents selected from: halogen, CF₃-、CF₃O-、CH₃O-、-C(O)OH、-C(O)OC_1-3Alkyl, -C (O) CH₃、-CN、-NH-、-NH₂、-O-、-OH、-NHCH₃、-N(CH₃)₂And C_1-3An alkyl group;

a. b and c are each independently an integer of 0,1, 2 or 3, provided that at least one of a, b or c is 1;

k and k' are each independently an integer of 0 or 1;

each p is independently an integer from 1 to 14;

each q is independently an integer from 1 to 12;

each AA is independently an amino acid;

each r is 1 to 12;

m is an integer of 1 to 4;

n is an integer from 1 to 4; and

the bond represents a single bond or a double bond.

In certain embodiments of the antibody-drug conjugate (ADC) of formula (Ib), R is selected from W, (L) as defined herein¹)_a、(L²)_b、(L³)_c、Z、W-(L¹)_a-(L²)_b-(L³)_c、(L¹)_a-(L²)_b-(L³)_c-Z, and W- (L)¹)_a-(L²)_b-(L³)_c-Z. In certain embodiments, R is selected from W, (L)¹)_a、(L²)_b、(L³)_cAnd W- (L)¹)_a-(L²)_b-(L³)_c. In certain embodiments, R is selected from Z, (L)¹)_a-(L²)_b-(L³)_c-Z and W- (L)¹)_a-(L²)_b-(L³)_c-Z。

In certain embodiments of the antibody-drug conjugate (ADC) of formula (Ib), R is a detectable probe. In certain embodiments, R is a fluorophore, chromophore, radiolabel, enzyme, ligand, antibody or antibody fragment. In certain embodiments, R is a ligand (e.g., a ligand specific for a receptor on a tumor cell, such as a prostate-specific membrane antigen, or a virus-infected cell, such as an HIV-infected cell).

In certain embodiments of the antibody-drug conjugates (ADCs) of formula (Ib), R is through an amide, N- (C)_1-6Alkyl) amides, carbamates, N- (C)_1-6Alkyl) carbamates, amines, N- (C)_1-6Alkyl) amines, ethers, thioethers, ureas, N- (C)_1-6Alkyl) urea, or N, N-di (C)_1-6Alkyl) urea linkage to the rest of the linker molecule.

In certain embodiments of the antibody-drug conjugates (ADCs) of formula (Ia) or (Ib), each L¹，L²And L³Independently selected from-NHC (O) -, -C (O) NH-, - (CH)₂CH₂O)_p、-(CH₂CH₂O)_pCH₂CH₂-、-CH₂CH₂-(CH₂CH₂O)_p-、-OCH(CH₂O-)₂、-(AA)_r-, unsubstituted phenyl and substituted by 1 or 2 substituents selected from the group consisting of halogen, CF3-, CF3O-, CH3O-, -C (O) OH, -C (O) OC_1-3Alkyl, -C (O) CH₃，-CN，-NH-，-NH2，-O-，-OH，-NHCH3，-N(CH₃)₂And C1-3 alkyl; wherein a, b and c are each independently 0 or 1; and each p and r is independently 1,2 or 3. In certain embodiments, L¹、L²And L³Is- (AA)_r-, wherein- (AA)_rIs ValCit (e.g., the first amino acid is valine, the second amino acid is citrulline, and r is 1). In certain embodiments, L¹、L²And L³ToOne or more is- (AA) r-, wherein- (AA)_rIs ValAla (e.g., the first amino acid is valine, the second amino acid is alanine, and r is 1). In certain embodiments, L¹、L²And L³Is one or more of-C (O) OH and-NH₂A substituted phenylene group. In certain embodiments, L¹、L²And L³is-C (O) O-and-NH-substituted phenylene. In certain embodiments, L¹、L²And L³Is-oc (o) -and-NH-substituted phenylene. In certain embodiments, L¹、L²And L³is-O-and-NH-substituted phenylene. In certain embodiments, L¹、L²And L³Is para-aminophenyl (PAB), which is optionally substituted with C (O) O-, -OC (O) -or-O-. In certain embodiments, L¹Is- (CH)₂)_q-，L²Is absent, L³Is absent, and CTX and (L)¹)_a-(L²)_b-(L³)_cLinked by an amide bond. In certain embodiments, L¹Is- (CH)₂)_q-，L²Is- (OCH)₂CH₂)_p-，L³Is absent, and CTX is bonded to (L) through an amide bond¹)_a-(L²)_b-(L³)_c. In certain embodiments, L¹Is- (CH)₂CH₂O)_p-，L²Is- (CH)₂)_q-, L3 is absent and CTX is bonded to (L) by an amide bond¹)_a-(L²)_b-(L³)_c. In certain embodiments, each L is¹Independently selected from- (CH)₂CH₂O)_pCH₂CH₂-and-CH₂CH₂-(CH₂CH₂O)_p-，L²Is absent, L³Is absent, and CTX is bonded to (L) through an amide bond¹)_a-(L²)_b-(L³)_c. In certain embodimentsEach of L¹Independently selected from- (CH)₂)_q-、-(CH₂CH₂O)_p、-(CH₂CH₂O)_pCH₂CH₂-、-CH₂CH₂-(CH₂CH₂O)_p-, and-C (O) -, L²Is Val-Cit, L³Is PAB and CTX is bonded to (L) through an amide bond¹)_a-(L²)_b-(L³)_c. In certain embodiments, each L is¹Independently selected from- (CH)₂)_q-、-(CH₂CH₂O)_p、-(CH₂CH₂O)_pCH₂CH₂-、-CH₂CH₂-(CH₂CH₂O)_p-, and-C (O) -, L²Is Val-Cit, L³Is PAB and CTX is bonded to (L) through an amide bond¹)_a-(L²)_b-(L³)_c. In certain embodiments, each L is¹Independently selected from- (CH)₂)_q-、-(CH₂CH₂O)_p、-(CH₂CH₂O)_pCH₂CH₂-、-CH₂CH₂-(CH₂CH₂O)_p-, and-C (O) -, L²Is Val-Ala, L³Is PAB and CTX is bonded to (L) through an amide bond¹)_a-(L²)_b-(L³)_c。

In certain embodiments of the antibody-drug conjugate (ADC) of formula (Ia) or (Ib), the CTX is selected from the group consisting of a tubulin stabilizing agent, a tubulin destabilizing agent, a DNA alkylating agent, a DNA minor groove binding agent, a DNA intercalating agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a gyrase inhibitor, a protein synthesis inhibitor, a proteosome inhibitor, and an antimetabolite.

In certain embodiments of the antibody-drug conjugate (ADC) of formula (Ia) or (Ib), the CTX is a chemotherapeutic agent. One of ordinary skill in the art will know of suitable chemotherapeutic agents, such as those disclosed in chu.e., Devite, v.t., 2012, physion' cancer chemistry Drug Manual 2012(Jones & Bartlett Learning Oncology) and the like.

In certain embodiments, CTX may be any FDA-approved chemotherapeutic agent. In certain embodiments, CTX may be any FDA-approved chemotherapeutic agent useful in the treatment of cancer.

In certain embodiments, the CTX is selected from the group consisting of alkylating agents, anthracyclines, cytoskeletal disruptors (taxanes), epothilones (epothilones), histone deacetylase inhibitors (HDACs), inhibitors of topoisomerase I, inhibitors of topoisomerase II, kinase inhibitors, monoclonal antibodies, nucleotide analogs, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids or derivatives thereof, and radioisotopes.

In certain embodiments, the CTX is selected from the group consisting of actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine (capecitabine), cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunomycin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone (epothilone), etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, Imatinib (Imatinib), irinotecan, methyldichloroethylamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed (pemetrexed), teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, doxine, and vinorelbine.

In certain embodiments, the CTX is selected from the group consisting of a tubulin stabilizing agent, a tubulin destabilizing agent, a DNA alkylating agent, a DNA minor groove binder, a DNA intercalator, a topoisomerase I inhibitor, a topoisomerase II inhibitor, a gyrase inhibitor, a protein synthesis inhibitor, a proteosome inhibitor, and an antimetabolite.

In certain embodiments, the CTX is selected from actinomycin D, amonafide, Auristatin (Auristatin), benzophenone, benzothiazole, calicheamicin, camptothecin, CC-1065(NSC298223), cimadrol, colchicine, combretastatin a4, dolastatin (dolastatin), doxorubicin, eletrinaGermany, Emtansine (DM1), Etoposide KF-12347 (Leinamycin), maytansinoids, methotrexate, mitoxantrone, nocodazole, proteosome inhibitor 1(PSI1), Styraponin A, T-2 toxin (trichothecene analog), paclitaxel, tubulin (tubulysin),

And vincristine. In certain embodiments, the CTX is an auristatin (auristatin), calicheamicin, maytansinoids, or tubulysin (tubulysin).

In certain embodiments, the CTX is monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), Pyrrolobenzodiazepine (PDB), calicheamicin γ, maytansine (mertansine), or tubulin T2. In certain embodiments, the CTX is MMAE or MMAF. In certain embodiments, the CTX is PDB. In certain embodiments, the CTX is tubulin T2. In certain embodiments, the CTX tubulin T3, or tubulin T4, the structure of which is provided below:

5.3 other molecules binding to BTN1A1 or BTN1A1 ligands

In another aspect, provided herein are molecules that selectively bind BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), whereby the molecules can inhibit binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the molecule is not an antibody and does not include an antigen binding domain. In some embodiments, the molecule is a decoy receptor, such as a GAL-1, GAL-9, NRP-2, BTLA or BTN1A1 decoy receptor or a soluble receptor. In some embodiments, the molecule can inhibit the formation of a BTN1a1-BTN1a1 ligand complex (e.g., a complex comprising GAL-1, GAL-9, NRP-2, or BTLA and BTN1a 1). In some embodiments, the molecule can disrupt the BTN1a1-BTN1a1 ligand complex formed (e.g., a complex comprising GAL-1, GAL-9, NRP-2, or BTLA and BTN1a 1). In some embodiments, provided herein are molecules that selectively bind BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) that can inhibit binding of BTN1a1 ligand to BTN1a1, and can inhibit the immunosuppressive function of BTN1a1 or BTN1a1-BTN1a1 ligand complex.

In another aspect, provided herein are molecules that selectively bind BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), whereby the molecules can inhibit binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the molecule is not an antibody and does not have an antigen binding fragment. In some embodiments, the molecule can inhibit binding of BTN1a1 to GAL-1. In some embodiments, the molecule may inhibit binding of BTN1a1 to GAL-9. In some embodiments, the molecule can inhibit binding of BTN1A1 to NRP-2. In some embodiments, the molecule can inhibit binding of BTN1a1 to BTLA. In some embodiments, the molecule may completely inhibit binding of BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, the molecule may at least partially inhibit binding of a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, the molecule can inhibit at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, binding of BTN1A1 to a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) or inhibition thereof is determined using surface plasmon resonance, biolayer interferometry, or co-immunoprecipitation. In some embodiments, the molecule is capable of inhibiting binding of BTN1a1 to a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) with an IC50 value of less than 1 μ Μ, less than 900nM, less than 800nM, less than 700nM, less than 600nM, less than 500nM, less than 400nM, less than 300nM, less than 200nM, less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 60nM, less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, or less than 1 nM. In some embodiments, the neutralization assay is a surface plasmon resonance, biolayer interferometry, co-immunoprecipitation, FRET or TR-FRET assay, or ELISA.

In some embodiments, the molecule selectively binds to BTN1a1, whereby the molecule can inhibit the binding of two or more BN1a1 ligands (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, the molecule may inhibit the binding of GAL-1 and GAL-9 to BTN1a 1. In some embodiments, the molecule may inhibit the binding of GAL-1 and NRP-2 to BTN1A 1. In some embodiments, the molecule can inhibit binding of GAL-1 and BTLA to BTN1a 1. In some embodiments, the molecule can inhibit the binding of GAL-9 and NRP-2 to BTN1A 1. In some embodiments, the molecule can inhibit binding of GAL-9 and BTLA to BTN1a 1. In some embodiments, the molecule can inhibit the binding of NRP-2 and BTLA to BTN1a 1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and NRP-2 to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and BTLA to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-1, NRP-2, and BTLA to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-9, NRP-2, and BTLA to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, NRP-2, and BTLA to BTN1A 1.

In some embodiments, the molecule selectively binds to the extracellular domain (ECD) of BTN1a 1.

In some embodiments, a molecule provided herein can bind a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) with a dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less or 1nM or less. In some embodiments, the molecule binds to GAL-1 with a dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less. In some embodiments, the molecule binds to Gal-9 with a dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less. In some embodiments, the molecule can bind to NRP-2 with a dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less. In some embodiments, the molecule can bind to BTLA with a dissociation constant of 1 μ Μ or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less or 1nM or less.

In some embodiments, the molecule may modulate the activity or signaling of BTN1a1, or modulate the activity or signaling of a complex of BTN1a1 and BTN1a1 ligands, such as GAL-1, GAL-9, NRP-2, or BTLA.

In some embodiments, the molecule can modulate T cell activity. In some embodiments, the T cell is a CD8+ cell. In some embodiments, the molecule can increase T cell activation or T cell proliferation. In some embodiments, the molecule can inhibit T cell apoptosis.

In some embodiments, the molecule is a GAL-1 decoy. In some embodiments, the molecule is a GAL-1 decoy as described in International patent application PCT/US2002/031273 (e.g., published as WO2003026494A3), which is incorporated herein by reference. In some embodiments, the molecule is a GAL-9 decoy. In some embodiments, the molecule is an NRP-2 bait. In some embodiments, the molecule is a BTLA decoy receptor. In some embodiments, the molecule is a soluble BTLA receptor (e.g., a BTLA extracellular domain construct, such as a BTLA-ECD-Fc construct). In some embodiments, the molecule is a membrane-bound BTLA decoy receptor (e.g., a truncated BTLA lacking a cytoplasmic domain). In some embodiments, the molecule is a BTN1a1 decoy or a soluble receptor. In some embodiments, the molecule is a BTN1a1 binding agent, as described in international application No. pct/US20104/071853 (e.g., published as WO2010100219a 1).

5.4 composition

Also provided herein are compositions having a molecule that selectively binds BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), whereby the molecule can inhibit binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the molecule is not an antibody and does not include an antigen binding fragment. In some embodiments, the molecule is a decoy receptor, such as a GAL-1, GAL-9, NRP-2, BTLA or BTN1A1 decoy receptor or a soluble receptor.

In another aspect, provided herein are compositions comprising molecules having antigen-binding fragments that immunospecifically bind to BTN1a1 or BTN1a1 ligand, whereby the molecules can inhibit the binding of BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN 1A. In some embodiments, the antigen binding fragment immunospecifically binds BTN1A (e.g., glycosylated BTN1A or BTN1A dimer). In some embodiments, the antigen binding fragment immunospecifically binds to a BTN1A ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA). In some embodiments, the antigen binding fragment immunospecifically binds to GAL-1. In some embodiments, the antigen binding fragment immunospecifically binds to GAL-9. In some embodiments, the antigen binding fragment immunospecifically binds to NRP-2. In some embodiments, the antigen binding fragment immunospecifically binds BTLA. In some embodiments, the antigen binding fragment immunospecifically binds BTN 1A. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at positions N55, N215, and/or N449. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1A glycosylated at position N55. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at position N215. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at position N449. In some embodiments, the antigen binding fragment immunospecifically binds to one or more glycosylation motifs. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at positions N55 and N215. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at positions N215 and N449. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at positions N55 and N449. In some embodiments, the antigen binding fragment immunospecifically binds to BTN1a1 glycosylated at positions N55, N215, and N449. In some embodiments, the antigen binding fragment immunospecifically binds to a BTN1a1 dimer, e.g., a BTN1a1 dimer that is glycosylated at one or more of positions N55, N215, and N449 of one or more BTN1a1 monomers in the BTN1A dimer.

In some embodiments, the compositions have a molecule with an antigen-binding fragment that immunospecifically binds to BTN1a1, whereby the molecule can inhibit binding of BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2 or B-and BTLA) to BTN 1A. In some embodiments, the molecule can inhibit binding of BTN1a1 to GAL-1. In some embodiments, the molecule may inhibit binding of BTN1a1 to GAL-9. In some embodiments, the molecule can inhibit binding of BTN1A1 to NRP-2. In some embodiments, the molecule can inhibit binding of BTN1a1 to BTLA. In some embodiments, the molecule can completely inhibit the binding of a BTN1A1 ligand (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A 1. In some embodiments, the molecule may at least partially inhibit binding of a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, the molecule can inhibit at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of BTN1a1 to a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA). In some embodiments, binding of BTN1a1 to a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) or inhibition thereof is determined using surface plasmon resonance, biolayer interferometry, or co-immunoprecipitation. In some embodiments, the molecule is capable of inhibiting binding of BTN1a1 to a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) with an IC50 value of less than 1 μ Μ, less than 900nM, less than 800nM, less than 700nM, less than 600nM, less than 500nM, less than 400nM, less than 300nM, less than 200nM, less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 60nM, less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, or less than 1 nM. In some embodiments, the neutralization assay is a surface plasmon resonance, biolayer interferometry, co-immunoprecipitation, FRET or TR-FRET assay, or ELISA.

In some embodiments, the compositions have a molecule with an antigen-binding fragment that immunospecifically binds to BTN1a1, whereby the molecule can inhibit the binding of two or more BN1a1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, the molecule may inhibit the binding of GAL-1 and GAL-9 to BTN1a 1. In some embodiments, the molecule may inhibit the binding of GAL-1 and NRP-2 to BTN1A 1. In some embodiments, the molecule can inhibit binding of GAL-1 and BTLA to BTN1a 1. In some embodiments, the molecule can inhibit the binding of GAL-9 and NRP-2 to BTN1A 1. In some embodiments, the molecule can inhibit binding of GAL-9 and BTLA to BTN1a 1. In some embodiments, the molecule may inhibit binding of NRP-2 and BTLA to BTN1a 1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and NRP-2 to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, and BTLA to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-1, NRP-2, and BTLA to BTN 1A. In some embodiments, the molecule can inhibit the binding of GAL-9, NRP-2, and BTLA to BTN 1A. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, NRP-2, and BTLA to BTN 1A.

In some embodiments, the compositions have a molecule that has an antigen-binding fragment that immunospecifically binds to a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), whereby the molecule is capable of inhibiting the binding of BTN1A ligand to BTN1a 1. In some embodiments, the antigen-binding fragment immunospecifically binds to GAL-1, and the molecule is capable of inhibiting the binding of GAL-1 to BTN1a 1. In some embodiments, the antigen-binding fragment immunospecifically binds to GAL-9, and the molecule is capable of inhibiting binding of GAL-9 to BTN1a 1. In some embodiments, the antigen-binding fragment immunospecifically binds to NRP-2, and the molecule is capable of inhibiting the binding of NRP-2 to BTN 1A. In some embodiments, the antigen binding fragment immunospecifically binds BTLA, and the molecule can inhibit binding of BTLA to BTN 1A. In some embodiments, the molecule may completely inhibit the binding of a BTN1A1 ligand (such as GAL-1, GAL-9, NRP-2, or BTLA) to BTN1A 1. In some embodiments, the molecule may at least partially inhibit binding of a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, the molecule can inhibit binding of at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of 1BTN1A ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, binding or inhibition of BTN1a1 to a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) is determined using surface plasmon resonance, biolayer interferometry, or co-immunoprecipitation. In some embodiments, the molecule is capable of inhibiting binding of BTN1a1 to a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) with an IC50 value of less than 1 μ Μ, less than 900nM, less than 800nM, less than 700nM, less than 600nM, less than 500nM, less than 400nM, less than 300nM, less than 200nM, less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 60nM, less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, or less than 1 nM. In some embodiments, the neutralization assay is a surface plasmon resonance, biolayer interferometry, co-immunoprecipitation, FRET or TR-FRET assay, or ELISA.

In some embodiments, provided herein are compositions having a molecule having an antigen-binding fragment that binds a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) with a dissociation constant of 1 μ Μ or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less, whereby the molecule is capable of inhibiting the binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the antigen-binding fragment can bind to GAL-1 with a dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less. In some embodiments, the antigen-binding fragment can bind to GAL-9 with a dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less. In some embodiments, the antigen-binding fragment can bind to NRP-2 with a dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less. In some embodiments, the antigen-binding fragment can bind to BTLA with a binding dissociation constant of 1 μ Μ or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less.

In some embodiments, the molecules in the compositions provided herein have an antigen-binding fragment that immunospecifically binds BTN1a1, wherein the antigen-binding fragment preferentially binds glycosylated BTN1a1 over non-glycosylated BTN1a 1. In some embodiments, glycosylated BTN1a1 is a BTN1a1 dimer. In some embodiments, the antigen binding fragment preferentially binds BTN1a1 glycosylated at positions N55, N215, and/or N449 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 glycosylated at position N55 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 glycosylated at position N215 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 glycosylated at position N449 over non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to one or more glycosylation motifs. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 glycosylated at positions N55 and N215 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 glycosylated at positions N215 and N449 over non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds BTN1a1 glycosylated at positions N55 and N449 over non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds BTN1a1 glycosylated at positions N55, N215, and N449 over non-glycosylated BTN1a 1.

In some embodiments, the molecules in the compositions provided herein have an antigen-binding fragment that immunospecifically binds BTN1a1, wherein the antigen-binding fragment preferentially binds BTN1a1 dimer relative to BTN1a1 monomer. In some embodiments, one or more of BTN1a1 monomers in the BTN1a1 dimer are glycosylated at one or more of positions N55, N215, and N449.

In some embodiments, the molecules in the compositions provided herein have an antigen binding fragment that binds to the K to which glycosylated BTN1a1 binds_DK exhibited in comparison to binding to unglycosylated BTN1A1_DLess than half. In some embodiments, the antigen binding fragment binds to K of glycosylated BTN1A1_DRelative to K exhibited in combination with unglycosylated BTN1A1_DAt least 2 times smaller. In some embodiments, the antigen binding fragment binds to K of glycosylated BTN1A1_DRelative to K exhibited in combination with unglycosylated BTN1A1_DAt least 5 times smaller. In some embodiments, the antigen binding fragment binds to K of glycosylated BTN1A1_DRelative to K exhibited in combination with unglycosylated BTN1A1_DAt least 10 times smaller. In some embodiments, the antigen binding fragment binds to K of glycosylated BTN1A1_DRelative to K exhibited in combination with unglycosylated BTN1A1_DAt least 15 times smaller. In some embodiments, the antigen binding fragment binds to K of glycosylated BTN1A1_DRelative to K exhibited in combination with unglycosylated BTN1A1_DAt least 20 times smaller. In some embodiments, the antigen binding fragment binds to K of glycosylated BTN1A1_DRelative to K exhibited in combination with unglycosylated BTN1A1_DAt least 25 times smaller. In some embodiments, the antigen binding fragment binds to K of glycosylated BTN1A1_DRelative to K exhibited in combination with unglycosylated BTN1A1_DAt least 30 times smaller. In some embodiments, the antigen binding fragment binds to K of glycosylated BTN1A1_DRelative to K exhibited in combination with unglycosylated BTN1A1_DAt least 40 times smaller. In some embodiments, the antigen binding fragment binds to K of glycosylated BTN1A1_DRelative to K exhibited in combination with unglycosylated BTN1A1_DAt least 50 times smaller.

In some embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRatio of exhibited K bound to BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DOne half smaller. In some embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRatio of K exhibited in combination with BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 2 times smaller. In some embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRatio of K exhibited in combination with BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 5 times smaller. In some embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRatio of K exhibited in combination with BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 10 times smaller. In some embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DThe ratio exhibits binding to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer)K of_DAt least 15 times smaller. In some embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRatio of K exhibited in combination with BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 20 times smaller. In some embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRatio of K exhibited in combination with BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 25 times smaller. In some embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) _DRatio of K exhibited in combination with BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 30 times smaller. In some embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRatio of K exhibited in combination with BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 40 times smaller. In some embodiments, the antigen binding fragment binds to K bound to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer)_DRatio of K exhibited in combination with BTN1A1 monomer (e.g., glycosylated BTN1A1 monomer)_DAt least 50 times smaller.

In some embodiments, the antigen binding fragment binds to glycosylated BTN1a1 at least twice as much MFI as non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment binds to glycosylated BTN1a1 at least 5-fold greater than MFI bound to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment binds to glycosylated BTN1a1 at least 10-fold greater than MFI bound to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment binds to glycosylated BTN1a1 at least 15-fold greater than MFI bound to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment binds to glycosylated BTN1a1 at least 20-fold greater than MFI bound to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment binds to glycosylated BTN1a1 at least 25-fold greater than MFI bound to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment binds to glycosylated BTN1a1 at least 30-fold greater than MFI bound to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment binds to glycosylated BTN1a1 at least 40-fold greater than MFI bound to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment binds to glycosylated BTN1a1 at least 50-fold greater than MFI bound to non-glycosylated BTN1a 1.

In some embodiments, the antigen binding fragment binds to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) at least twice as high as the MFI exhibited by a BTN1a1 monomer (e.g., a glycosylated BTN1A monomer). In some embodiments, the antigen binding fragment binds to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) with an MFI at least 5-fold higher than that exhibited by a BTN1a1 monomer (e.g., a glycosylated BTN1A monomer). In some embodiments, the antigen binding fragment binds to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) with an MFI at least 10-fold higher than the MFI exhibited by a BTN1a1 monomer (e.g., a glycosylated BTN1A monomer). In some embodiments, the antigen binding fragment binds to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) with an MFI at least 15-fold higher than that exhibited by a BTN1a1 monomer (e.g., a glycosylated BTN1A monomer). In some embodiments, the antigen binding fragment binds to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) with an MFI at least 20-fold higher than that exhibited by a BTN1a1 monomer (e.g., a glycosylated BTN1A monomer). In some embodiments, the antigen binding fragment binds to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) with an MFI at least 25-fold higher than that exhibited by a BTN1a1 monomer (e.g., a glycosylated BTN1A monomer). In some embodiments, the antigen binding fragment binds to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) with an MFI at least 30-fold greater than the MFI exhibited by a BTN1a1 monomer (e.g., a glycosylated BTN1A monomer). In some embodiments, the antigen binding fragment binds to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) with an MFI at least 35-fold greater than the MFI exhibited by a BTN1a1 monomer (e.g., a glycosylated BTN1A monomer). In some embodiments, the antigen binding fragment binds to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) with an MFI at least 40-fold greater than the MFI exhibited by a BTN1a1 monomer (e.g., a glycosylated BTN1A monomer). In some embodiments, the antigen binding fragment binds to a BTN1A dimer (e.g., a glycosylated BTN1A dimer) with an MFI at least 50-fold greater than the MFI exhibited by a BTN1a1 monomer (e.g., a glycosylated BTN1A monomer).

In another aspect, provided herein are compositions having a molecule with an antigen-binding fragment immunospecifically masking BTN1a1 glycosylation at positions N55, N215, and/or N449. In certain embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N55. In certain embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N215. In certain embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N449. In certain embodiments, the antigen binding fragment immunospecifically masks one or more glycosylation motifs of BTN1a 1. In certain embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at positions N55 and N215. In certain embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at positions N215 and N449. In certain embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at positions N55 and N449. In certain embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at positions N55, N215, and N449.

In some embodiments, the composition may have at least 0.1% by weight of an antibody or other molecule described herein. In some embodiments, the composition can have at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7% 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more by weight of an anti-BTN 1a1 antibody, an anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) antibody, or other molecule having an antigen binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand. In other embodiments, for example, the anti-BTN 1a1 antibody, anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), or other molecule having an antigen binding fragment that immunospecifically binds to BTN1a1 may comprise about 2% to about 75%, about 25% to about 60%, about 30% to about 50%, or any range therein, by weight of the composition.

The composition may be a pharmaceutical composition having as an active ingredient an anti-BTN 1a1 antibody, an anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-or BTLA) antibody, or other antigen binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand, and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise one or more additional active ingredients. The pharmaceutically acceptable carrier may be a carrier approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia, european pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The preparation of pharmaceutical compositions having as an active ingredient an antibody or other molecule as described herein is known to those skilled in the art in light of the present disclosure, as exemplified in Remington's pharmaceutical sciences, 18 th edition, 1990, which is incorporated herein by reference. Moreover, for animal (including human) administration, it is understood that the formulations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA office of biological standards.

The pharmaceutically acceptable carrier includes a liquid, a semi-solid, i.e., a paste, or a solid carrier. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. The pharmaceutically acceptable carrier may include aqueous solvents (e.g., water, alcohol/aqueous solutions, ethanol, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, and the like), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases, parabens (e.g., methyl paraben, propyl paraben), chlorobutanol, phenol, sorbic acid, thimerosal), isotonic agents (e.g., sugars, sodium chloride), absorption delaying agents (e.g., aluminum monostearate, gelatin), salts, drugs, drug stabilizers (e.g., buffers, amino acids such as glycine and lysine, carbohydrates such as glucose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, and the like), gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorants, dyes, liquids, and nutrient supplements, such materials, and combinations thereof, as will be known to those of ordinary skill in the art. Unless any conventional vehicle, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in an administrable composition for practicing the present methods is appropriate. The pH and exact concentration of the various ingredients in the pharmaceutical composition are adjusted according to well-known parameters. In accordance with certain aspects of the present disclosure, the compositions may be combined with the carrier in any convenient and practical manner, i.e., by dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, grinding, or the like. Such procedures are conventional to those skilled in the art.

In some embodiments, the pharmaceutically acceptable carrier may be a pH buffered aqueous solution. Examples include buffers such as phosphate, citrate and other organic acids; antioxidants include ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN^TMPolyethylene glycol (PEG) and PLURONICS^TM。

In some embodiments, the pharmaceutically acceptable carrier can be a sterile liquid, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water may be the carrier, particularly when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, calcium carbonate, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, polysorbate-80 and the like. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.

The anti-BTN 1a1 antibody, anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, RP-or BTLA) antibody, or other molecule having an antigen binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand, can be formulated into a composition in free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, for example with the free amino groups of the protein composition, or with inorganic acids, for example hydrochloric or phosphoric acids, or with organic acids, for example acetic, oxalic, tartaric or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium, ammonium, calcium or iron; or organic base such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine.

In further embodiments, provided herein are pharmaceutical compositions having lipids. Lipids may broadly include a class of substances characterized by being insoluble in water and extractable with organic solvents. Examples include compounds containing long chain aliphatic hydrocarbons and derivatives thereof. Lipids may be naturally occurring or synthetic (i.e., designed or produced by humans). The lipid may be a biological substance. Biolipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysophospholipids, glycosphingolipids, glycolipids, sulfates, lipids with ether and ester linked fatty acids, polymerizable lipids, and combinations thereof. Compounds other than those specifically described herein, which are understood to be lipids by those skilled in the art, may also be used.

One of ordinary skill in the art will be familiar with the range of techniques that can be used to disperse the composition in a lipid carrier. For example, the antibody may be dispersed in a solution containing a lipid, solubilized with a lipid, emulsified with a lipid, mixed with a lipid, associated with a lipid, covalently bound to a lipid, contained as a lipid suspension in a lipid, comprised or complexed with micelles or liposomes, or associated with a lipid or lipid structure by any method known to one of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

Typically, the components of the compositions are either supplied separately or mixed together in unit dosage form, e.g., as a lyophilized powder or anhydrous concentrate in a closed container such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water or saline for injection may be provided to mix the ingredients prior to administration.

The amount of active ingredient in each therapeutically useful composition can be prepared in such a way as to obtain a suitable dosage in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will occur to those skilled in the art and thus various dosages and treatment regimens are required.

A unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a pharmaceutical composition calculated to produce the desired response discussed above in association with its administration, i.e., the appropriate route and treatment regimen. Depending on the number of treatments and the unit dose, the amount to be administered depends on the desired effect. The number of reagent doses of the composition of this embodiment administered to a patient or subject can be determined by physical and physiological factors such as weight, age, health, sex of the subject, type of disease to be treated, extent of disease penetration, previous or concurrent therapeutic intervention, self-morbidity of the patient, route of administration, and potency, stability and toxicity of the particular therapeutic substance. In other non-limiting examples, the dose can be about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 milligram/kg/body weight or higher per administration, and any range derivable therein. In non-limiting examples of ranges derivable from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 micrograms/kg/body weight to about 500 milligrams/kg/body weight, and the like may be used in accordance with the numbers described above. In any case, the physician responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for an individual subject.

One of ordinary skill in the art will appreciate that the compositions described herein are not limited by the particular nature of the therapeutic article. For example, such compositions may be provided in formulations with physiologically tolerable liquid, gel or solid carriers, diluents and excipients. These therapeutic articles can be administered to mammals for veterinary use, e.g., for livestock, and clinical use in humans in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy varies according to the type and mode of administration employed, as well as the particular needs of the individual subject. The actual dosage amount of the composition to be administered to an animal patient, including a human patient, can be determined by physical and physiological factors, such as, for example, body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic intervention, patient morbidity, and the route of administration. Depending on the dose and route of administration, the preferred dose and/or the number of administrations of the effective amount may vary depending on the response of the subject. In any case, the physician responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for an individual subject.

5.5 therapeutic uses and methods of treatment

BTN1a1 is specific and highly expressed in cancer cells.

In another aspect, provided herein is a therapeutic use of a molecule that selectively binds BTN1a1 or a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) in the treatment of cancer, wherein the molecule is capable of inhibiting the binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the molecule is not an antibody and does not include an antigen binding fragment. In some embodiments, the molecule is a decoy receptor, such as a GAL-1, GAL-9, NRP-2, BTLA or BTN1A1 decoy receptor or soluble receptor.

In another aspect, provided herein is a therapeutic use of a molecule having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) in the treatment of cancer, whereby the molecule can inhibit binding of BTN1a1 ligand to BTN1a 1. In some embodiments, these molecules bind to cancer cells expressing BTN1a1 and induce an immune response that results in the destruction of these cancer cells. In some embodiments, the molecule inhibits a BTN1a1-BTN1a1 ligand (e.g., GAL-1, GAL-2, NRP-2, BTLA) complex. For example, in some embodiments, the molecule may prevent the formation of a BTN1a1-BTN1a1 ligand complex or disrupt an already formed BTN1a1-BTN1a1 ligand complex. The molecules provided herein can enhance T cell-dependent apoptosis and inhibit proliferation of cancer cells.

In another aspect, the invention provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a molecule that selectively binds to BTN1a1 or a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), whereby the molecule inhibits binding of the BTN1a1 ligand to BTN1a 1. In some embodiments, the molecule is not an antibody and does not include an antigen binding fragment. In some embodiments, the molecule is a decoy receptor, such as a GAL-1, GAL-9, NRP-2, BTLA or BTN1A1 decoy receptor or soluble receptor.

In another aspect, the invention provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a molecule having an antigen binding fragment that immunospecifically binds BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), whereby the molecule inhibits binding of BTN1a1 ligand BTN1a 1.

In some embodiments, the molecule has an antigen-binding fragment that immunospecifically binds to BTN1a1, whereby the molecule can inhibit binding of BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2 or B-and BTLA) to BTN1a 1. In some embodiments, the molecule can inhibit binding of BTN1a1 to GAL-1. In some embodiments, the molecule can inhibit binding of BTN1a1 to GAL-9. In some embodiments, the molecule can inhibit binding of BTN1A1 to NRP-2. In some embodiments, the molecule can inhibit binding of BTN1a1 to BTLA. In some embodiments, the molecule may completely inhibit binding of BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, the molecule may at least partially inhibit binding of a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, the molecule can inhibit at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, binding or inhibition of BTN1A1 to a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) is determined using surface plasmon resonance biolayer interferometry or co-immunoprecipitation. In some embodiments, the molecule is capable of inhibiting the binding of a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a1 with an IC50 value of less than 1 μ M, less than 900nM, less than 800nM, less than 700nM, less than 600nM, less than 500nM, less than 400nM, less than 300nM, less than 200nM, less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 60nM, less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, or less than 1 nM. In some embodiments, the neutralization assay is surface plasmon resonance, biolayer interferometry, co-immunoprecipitation, a FRET or TR-FRET assay, or ELISA.

In some embodiments, the molecule has an antigen-binding fragment that immunospecifically binds to BTN1a1, whereby the molecule can inhibit the binding of two or more BTN1a1 ligands (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, the molecule may inhibit the binding of GAL-1 and GAL-9 to BTN1a 1. In some embodiments, the molecule can inhibit the binding of GAL-1 and NRP-2 to BTN1A 1. In some embodiments, the molecule can inhibit binding of GAL-1 and BTLA to BTN1a 1. In some embodiments, the molecule can inhibit the binding of GAL-9 and NRP-2 to BTN1A 1. In some embodiments, the molecule can inhibit binding of GAL-9 and BTLA to BTN1a 1. In some embodiments, the molecule may inhibit binding of NRP-2 and BTLA to BTN1a 1. In some embodiments, the molecule may inhibit the binding of GAL-1, GAL-9 and NRP-2 to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9 and BTLA to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-1, NRP-2, and BTLA to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-9, NRP-2, and BTLA to BTN1A 1. In some embodiments, the molecule can inhibit the binding of GAL-1, GAL-9, NRP-2, and BTLA to BTN1A 1.

In some embodiments, the molecule has an antigen binding fragment that immunospecifically binds to a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), whereby the molecule is capable of inhibiting binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the antigen-binding fragment immunospecifically binds to GAL-1, and the molecule is capable of inhibiting binding of GAL-1 to BTN1a 1. In some embodiments, the antigen-binding fragment immunospecifically binds to GAL-9, and the molecule is capable of inhibiting binding of GAL-9 to BTN1a 1. In some embodiments, the antigen-binding fragment immunospecifically binds to NRP-2, and the molecule can inhibit binding of NRP-2 to BTN1a 1. In some embodiments, the antigen binding fragment immunospecifically binds BTLA, and the molecule is capable of inhibiting BTLA binding to BTN 1A. In some embodiments, the molecule may completely inhibit binding of BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, the molecule may at least partially inhibit binding of a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, the molecule can inhibit at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a 1. In some embodiments, binding of BTN1A1 to a BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) or inhibition thereof is determined using surface plasmon resonance, biolayer interferometry, or co-immunoprecipitation. In some embodiments, the molecule is capable of inhibiting the binding of a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) to BTN1a1 with an IC50 value of less than 1 μ M, less than 900nM, less than 800nM, less than 700nM, less than 600nM, less than 500nM, less than 400nM, less than 300nM, less than 200nM, less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 60nM, less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, or less than 1 nM. In some embodiments, the neutralization assay is a surface plasmon resonance, biolayer interferometry, co-immunoprecipitation, FRET or TR-FRET assay, or ELISA.

In some embodiments, the molecule has an antigen-binding fragment that binds a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) with a dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less, whereby the molecule is capable of inhibiting the binding of a BTN1a1 ligand to BTN1a 1. In some embodiments, the antigen-binding fragment can bind to GAL-1 with a dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less. In some embodiments, the antigen-binding fragment can bind to GAL-9 with a dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less. In some embodiments, the antigen-binding fragment can bind to NRP-2 with a binding dissociation constant of 1 μ M or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less. In some embodiments, an antigen-binding fragment can bind to BTLA with a binding dissociation constant of 1 μ Μ or less, 900nM or less, 800nM or less, 700nM or less, 600nM or less, 500nM or less, 400nM or less, 300nM or less, 200nM or less, 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, 10nM or less, 5nM or less, 3nM or less, or 1nM or less.

In some embodiments, the molecules provided herein have antigen binding fragments that immunospecifically bind to BTN1a1, including anti-BTN 1a1 antibodies that can cause internalization of BTN1a1 into lysosomes. Thus, also provided herein are methods of using the molecules provided herein to deliver a compound to a cell expressing BTN1a1 by contacting the cell with the molecules provided herein conjugated to the compound. The compound may be an imaging agent, therapeutic agent, toxin or radionuclide as described herein. The compounds may be conjugated to anti-BTN 1a1 antibodies. The conjugate can be any conjugate described herein, such as an ADC. The cell may be a cancer cell. The cell may also be a population of cells that includes both cancer cells and normal cells. Because cancer cells specifically and highly express BTN1a1, the molecules described herein can be used to achieve specific drug delivery to cancer cells, but not normal cells.

In some embodiments, the molecules provided herein, including anti-BTN 1a1 antibodies and anti-BTN 1a1 ligand (GAL-1, GAL-9, NRP-2, BTLA) antibodies, may modulate an immune response in a subject. In some embodiments, the molecule can promote T cell activation. In some embodiments, the molecule can promote T cell proliferation. In some embodiments, the molecule can increase cytokine production. In some embodiments, the molecules provided herein may also enhance T cell-dependent apoptosis of cells expressing BTN1a1 or inhibit proliferation of cells expressing BTN1a 1.

Accordingly, provided herein are methods of modulating an immune response in a subject by administering an effective amount of a molecule provided herein having an antigen-binding fragment that immunospecifically binds BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), capable of inhibiting the binding of BTN1a1 ligand to BTN1a1, including an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand antibody. Modulating an immune response may include (a) increasing T cell activation (e.g., CD 8)⁺T cell activation); (b) increase T cell proliferation; and/or (c) increase cytokine production. In some embodiments, the method further comprises administering an anti-PD 1 therapy or an anti-PD-L1 therapy.

Also provided herein are methods of enhancing T cell-dependent apoptosis in BTN1a 1-expressing cells by contacting the cells with an effective amount of a molecule described herein that has an antigen-binding fragment, including an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand antibody, that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule can inhibit binding of BTN1a1 ligand to BTN1a 1. Also provided herein are methods of inhibiting proliferation of a cell expressing BTN1a1 by contacting the cell with an effective amount of a molecule described herein having an antigen-binding fragment, including an anti-BTN 1a1 antibody, that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule can inhibit binding of BTN1a1 ligand to BTN1a 1. The cell may be a cancer cell. In some embodiments, the method further comprises administering an anti-PD 1 therapy or an anti-PD-L1 therapy.

In some embodiments, these molecules may be used to treat cancer by inhibiting the inhibitory activity of BTN1a1 in T cell activation or proliferation. Thus, provided herein are uses of these molecules in up-regulating the immune system of a subject by inhibiting or blocking BTN1a1 signaling. In some embodiments, provided herein are uses of these molecules to block BTN1a1 binding to T cells.

In some embodiments, these molecules cause the destruction of cancer cells through ADCC or CDC mechanisms. In some embodiments, these molecules are designed to have enhanced ADCC activity. In some embodiments, these molecules are engineered to have enhanced CDC activity. For example, these molecules can be designed to have enhanced interactions with Fc receptor-bearing killer cells. Methods of producing such engineered molecules are described herein, and are also known in the art.

In another aspect, the invention provides a method of killing or inhibiting proliferation of a cancer cell resistant to anti-PD-1 therapy or anti-PD-L1 therapy, comprising contacting the cell with an effective amount of a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA), whereby the molecule can inhibit binding of BTN1a1 or BTN1a1 ligand.

In some embodiments, provided herein is the use of a molecule having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein the molecule can inhibit the binding of BTN1a1 ligand to BTN1a1, including anti-BTN 1a1 antibodies and anti-BTN 1a1 ligand antibodies, in the treatment of a disease or disorder in a subject that overexpresses BTN1a 1. In some embodiments, the expression level of BTN1a1 in the subject is higher than the reference level. The reference level may be an average or intermediate expression level of BTN1a1 in a population of healthy individuals. The reference level may also be determined by statistically analyzing the expression level of a population of samples.

In another aspect, the invention provides a method of treating a cancer resistant or refractory to an anti-PD-1 therapy or an anti-PD-L1 therapy in a subject, comprising administering to the subject a therapeutically effective amount of a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule can inhibit binding of BTN1a1 ligand to BTN1a 1.

In some embodiments, the subject has a cancer that is resistant to anti-PD-1 therapy or anti-PD-L1 therapy. In some embodiments, the subject has a cancer resistant to a PD-1 therapy. In some embodiments, the subject has a cancer resistant to PD-L1 therapy.

In some embodiments, the subject has a cancer refractory to anti-PD-1 therapy or anti-PD-L1 therapy. In some embodiments, the subject has a cancer refractory to an anti-PD-1 therapy. In some embodiments, the subject has a cancer refractory to an anti-PD-L1 therapy.

In some embodiments, the anti-PD-1 therapy or anti-PD-L1 therapy includes an anti-PD-1 or anti-PD-L1 antibody or antibody fragment, or a soluble PD-1 or PD-L1 ligand, or an Fc-fusion protein thereof (e.g., AMP-224, PD-L2Fc fusion soluble receptor).

In some embodiments, the anti-PD-1 therapy comprises nivolumab (opdivo), pembrolizumab

Pidilizumab, AMP-514, or AMP-224.

In some embodiments, the anti-PD-1 treatment comprises an anti-PD-1 antibody provided in international application PCT/US 20126/64394.

In some embodiments, the anti-PD-L1 treatment comprises yw243.55.s70, MPDL3280A, MEDI-4736, MSB-0010718C or MDX-1105.

In some embodiments, the anti-PD-L1 treatment comprises an antibody provided in international application No. pct/US2016/024691, disclosed as WO2016/160792a1, and international application No. pct/US 2017/024027.

In some embodiments, the subject is treated for the first time (e.g., the subject has not received any anti-cancer therapy) prior to treatment with the molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). In some embodiments, the subject has received one or more anti-cancer treatments (e.g., chemotherapy, radiation therapy, surgery, or with another targeted anti-cancer drug, e.g., chemotherapy) in addition to anti-PD-1 therapy or anti-PD-L1 therapy prior to treatment with a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA)

(trastuzumab) therapy). In some embodiments, the subject received one or more anti-PD 1 therapies or anti-PD-L1 therapies prior to treatment with a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA).

In certain embodiments, the anti-PD-1 therapy or anti-PD-L1 therapy resistant or refractory cancer is a lung cancer or a breast cancer. In certain embodiments, the anti-PD-1 therapy or anti-PD-L1 therapy resistant or refractory cancer is a lung cancer. In certain embodiments, the anti-PD-1 therapy or anti-PD-L1 therapy resistant or refractory cancer is a breast cancer. In certain embodiments, the lung cancer is Lewis lung cancer. In certain embodiments, the breast cancer is breast cancer.

In some embodiments, a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) is administered parenterally. In some embodiments, the molecule comprises an anti-BTN 1a1 dimer antibody or antigen binding fragment thereof.

In some embodiments, the treatment produces at least one therapeutic effect, such as reducing the size of a tumor, reducing the number of metastatic lesions over time, a complete response, a partial response, or a stable disease.

In another aspect, provided herein is a method of treating cancer, comprising (i) obtaining a sample comprising cancer cells from a subject having cancer; (ii) determining the level of BTN1A1 or BTN1A1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in the sample; (iii) (iii) if the level of BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in the sample is greater than or equal to a reference level of BTN1a1 or BTN1a1 ligand, diagnosing that the subject is likely to be responsive to treatment with a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), and (iv) administering to the subject a therapeutically effective amount of the molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule inhibits binding of BTN1a1 ligand to BTN1a 1.

In another aspect, provided herein is a method of treating cancer, comprising (i) obtaining a sample comprising cancer cells from a subject having cancer; (ii) determining the level of PD-L1 in the sample; (iii) (iii) diagnosing that the subject is likely to be responsive to treatment with a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) that inhibits binding of BTN1a1 ligand to BTN1a1 if the level of PD-L1 in the sample is less than or equal to the reference level of PD-L1, and (iv) administering to the subject a therapeutically effective amount of a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand.

In another aspect, provided herein is a method of treating a cancer resistant or refractory to an anti-PD 1 therapy or an anti-PD-L1 therapy, comprising (i) obtaining a sample comprising cancer cells from a subject having a cancer resistant or refractory to an anti-PD 1 therapy or an anti-PD-L1 therapy; (ii) determining the level of BTN1A1 or BTN1A1 ligand (GAL-1, GAL-9, NRP-2, BTLA) in the sample; (iii) (iii) if the BTN1a1 or BTN1a1 ligand level in the sample is greater than or equal to the reference level of BTN1a1, diagnosing that the subject is likely to be responsive to treatment with a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), which molecule inhibits binding of BTN1a1 ligand to BTN1a1, and (iv) administering to the subject a therapeutically effective amount of the molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand.

In another aspect, provided herein is a method of treating a cancer resistant or refractory to anti-PDL therapy or anti-PD-L1 therapy, comprising (i) obtaining a sample comprising cancer cells from a subject having a cancer resistant or refractory to anti-PD 1 therapy or anti-PD-L1 therapy; (ii) determining the level of BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) and/or PD-L1 in the sample; (iii) (iii) diagnosing that the subject is likely to be reactive to treatment with a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) that inhibits binding of BTN1a1 ligand to BTN1a1 if the level of BTN1a1 or BTN1a1 ligand in the sample is greater than or equal to the reference level of BTN1a1 or BTN1a1 ligand and/or if the level of PD-L1 in the sample is equal to or less than the reference level of PD-L1, and (iv) administering to the subject a therapeutically effective amount of the molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand.

In another aspect, provided herein is a method of treating cancer, comprising (i) obtaining a sample comprising cancer cells from a subject having cancer; (ii) determining the level of BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) and/or PD-L1 in the sample; (iii) (iii) diagnosing that the subject is likely to be responsive to treatment with a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand, if the level of BTN1a1 or BTN1a1 ligand in the sample is greater than or equal to a reference level of BTN1a1 and/or if the level of PD-L1 in the sample is equal to or less than a reference level of PD-L1, whereby the molecule can inhibit binding of BTN1a1 to BTN1a1 ligand, and (iv) administering to the subject a therapeutically effective amount of the molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand.

In certain embodiments, the subject has a cancer resistant or refractory to anti-PD-1 therapy or anti-PD-L1 therapy. In certain embodiments, the subject has a cancer that is resistant to anti-PD-1 therapy or anti-PD-L1 therapy. In certain embodiments, the subject has a cancer resistant to an anti-PD-1 therapy. In certain embodiments, the subject has a cancer resistant to PD-L1 therapy. In certain embodiments, the subject has a cancer refractory to anti-PD-1 therapy or anti-PD-L1 therapy. In certain embodiments, the subject has a cancer refractory to an anti-PD-1 therapy. In certain embodiments, the subject has a cancer refractory to anti-PD-L1 therapy.

In certain embodiments, the subject has a cancer that is at least partially responsive to anti-PD-1 therapy or anti-PD-L1 therapy.

In certain embodiments, the BTN1a1 is expressed in the cancer. In certain embodiments, the BTN1a 1-expressing cancer includes, for example, breast cancer, neuroendocrine prostate cancer (NEPC), diffuse large B-cell lymphoma, melanoma, cancer from the national cancer institute cancer panel (NCI 60), uveal melanoma, pancreatic cancer, ovarian cancer, uterine cancer, lung adenocarcinoma, desmoplastic small round cell tumor, bladder cancer, colorectal cancer, lung squamous cell cancer, liver cancer, lung cancer, stomach cancer, cholangiocellular carcinoma, esophageal squamous cell cancer, head and neck cancer, sarcoma, prostate cancer, liver cancer, pancreatic cancer, pheochromocytoma or paraganglioma (PCPG), cervical cancer, glioma, or Acute Myelogenous Leukemia (AML).

In some embodiments, the method comprises determining the level of BTN1a1 or BTN1a1 in the sample. In some embodiments, the method comprises determining the level of PD-L1 in the sample. In some embodiments, the methods comprise determining the level of BTN1a1 or BTN1a1 ligand and PD-L1 in the sample.

In some embodiments, if the level of BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in the sample is greater than or equal to a reference level of BTN1a1 or BTN1a1 ligand, then diagnosing that the subject is likely to be reactive to molecular therapy comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule may inhibit binding of BTN1a1 ligand to BTN1a 1. In some embodiments, if the level of BTN1a1 or BTN1a1 ligand in the sample is higher than the reference level of BTN1a1 or BTN1a1 ligand, then diagnosing that the subject is likely to be responsive to molecular therapy comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand.

In some embodiments, the diagnostic subject may be responsive to treatment with a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein the molecule may inhibit binding of BTN1A ligand to BTN1a1, if the level of PD-L1 in the sample is less than or equal to the reference level of PD-L1. In some embodiments, if the level of PD-L1 in the sample is below the reference level of PD-L1, the diagnostic subject may be responsive to molecular therapy comprising an antigen binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand.

In some embodiments, if the level of BTN1a1 or BTN1A ligand in the sample is equal to or higher than the reference level of BTN1a1 and the level of PD-L1 is lower than or equal to the reference level of PD-L1, the diagnostic subject may be reactive to treatment with a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), which molecule may inhibit binding of BTN1a1 ligand to BTN1a 1. In some embodiments, if the level of BTN1a1 or BTN1A ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in the sample is greater than or equal to a reference level of BTN1a1 and the level of PD-L1 is less than a reference level of PD-L1, the diagnostic subject may be reactive to molecular therapy that includes an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), which molecule may inhibit binding of BTN1a1 ligand to BTN1a 1. In some embodiments, if the level of BTN1a1 or BTN1A ligand in the sample is above a reference level of BTN1a1, and the level of PD-L1 is below a reference level of PD-L1, the diagnostic subject may be reactive to a molecular therapy comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), which molecule may inhibit binding of BTN1a1 ligand to BTN1a 1.

The sample may be any solid or liquid sample from a subject.

In certain embodiments, the sample is a liquid biopsy sample. In certain embodiments, the sample used in the methods provided herein comprises a bodily fluid from a subject. Non-limiting examples of bodily fluids include blood (e.g., whole blood), plasma, amniotic fluid, aqueous humor, bile, cerumen, cowper's fluid, pre-ejaculatory fluid, chyle, chyme, female ejaculate, interstitial fluid, lymph, menses, milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, tears, urine, vaginal lubrication, vomit, water, stool, internal bodily fluids (including cerebrospinal fluid around the brain and spinal cord), synovial fluid, intracellular fluid (fluid inside the cell), and vitreous (fluid in the eyeball). In certain embodiments, the sample is a blood sample. The blood sample may be obtained using conventional techniques, such as described in Innis et al, eds., PCR Protocols (academic Press, 1990). Leukocytes can be isolated from blood samples using conventional techniques or commercially available kits, such as the RosetteSep kit (Stein Cell Technologies, Vancouver, Canada). Subpopulations of leukocytes, e.g., monocytes, B cells, T cells, monocytes, granulocytes, or lymphocytes, can be further isolated using conventional techniques, such as Magnetic Activated Cell Sorting (MACS) (Miltenyi Biotec, Auburn, California) or Fluorescence Activated Cell Sorting (FACS) (Becton Dickinson, San Jose, California).

In certain embodiments, the sample is a solid biopsy sample. In certain embodiments, the sample used in the current methods comprises a biopsy (e.g., a tumor biopsy). The biopsy may be from any organ or tissue, for example, skin, liver, lung, heart, colon, kidney, bone marrow, teeth, lymph nodes, hair, spleen, brain, breast, or other organ. Any biopsy technique known to those skilled in the art may be used to isolate a sample from a subject, for example, an open biopsy, a closed biopsy, a core biopsy, a resection biopsy, an excisional biopsy, or a fine needle biopsy.

In some embodiments, the sample is a paraffin-embedded formaldehyde-fixed tissue sample. In some embodiments, the sample is a tissue section. In some embodiments, the sample is provided on a tissue array.

In some embodiments, the level of the biomarker is measured by determining the protein level of the biomarker.

The level of BTN1a1, BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), or PD-L1 in a sample can be analyzed using any method known in the art. See, e.g., section 5.7 (companion diagnostics). In some embodiments, determining the level of BTN1a1, BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), or PD-L1 in a sample may comprise analyzing the nucleic acid level of BTN1a1, BTN1a1 ligand, or PD-L1 or the protein level of BTN1a1, BTN1a1 ligand, or PD-L1. The nucleic acid level of BTN1a1, BTN1a1 ligand, or PD-L1 can be analyzed, for example, using Polymerase Chain Reaction (PCR) methods (e.g., RT-PCR or Q-PCR), nucleic acid array-based methods (e.g., gene chips), or nucleic acid sequencing methods such as next generation sequencing methods. BTN1A1, BTN1A1 ligand or PD-L1 protein level can be determined using, for example, Western-Blot, ELISA, FACS, immunohistochemistry. BTN1a1, BTN1a1 ligand, or PD-L1 levels can be determined in an absolute quantitative manner (e.g., BTN1a1, BTN1a1 ligand, or PD-L1 weight per weight part of tissue, or molar amount per tissue volume or per liquid sample volume). In some embodiments, BTN1a1, BTN1a1 ligand, or PD-L1 levels are determined in a relative or semi-quantitative manner (e.g., relative intensities of BTN1a1 or PD-L1 specific staining in different regions of a tissue sample). BTN1a1, BTN1a1 ligand, or PD-L1 levels can be independently determined using the same method or different methods. In some embodiments, relative BTN1a1, BTN1a1 ligand, or PD-L1 levels are determined in tissue sections of solid tumor samples using immunohistochemistry in combination with fluorescence microscopy or bright field microscopy.

In some embodiments, determining the level of BTN1a1, BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), and/or PD-L1 in a sample comprises analyzing the expression of cell surface BTN1a1, BTN1a1 ligand, and/or PD-L1, e.g., using FACS assays or immunocytochemistry.

In certain embodiments, the treatment produces at least one therapeutic effect, e.g., a decrease in tumor size, a decrease in the number of metastatic lesions over time, a complete response, a partial response, or a stable disease.

In some embodiments, a reference is prepared using a control sample obtained from the subject prior to administering to the subject a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), the control sample being from the same source as the sample. In certain embodiments, a reference is prepared using a control sample obtained from a healthy subject not having cancer, the control sample being from the same source as the sample. In certain embodiments, a reference is prepared using a control sample obtained from a group of healthy subjects that do not have cancer, the control sample being from the same source as the sample. In certain embodiments, a reference is prepared using a control sample obtained from a second subject having cancer, the control sample being from the same source as the sample. In certain embodiments, a reference is prepared using a control sample obtained from a group of subjects having cancer, the control sample being from the same source as the sample.

5.4.1 diseases and disorders

In some embodiments, provided herein is the use of an antibody or other molecule to mediate increased production of a cytokine, such as IFN- γ. Accordingly, provided herein are uses of such antibodies or other molecules in the treatment of diseases and disorders that can be treated with cytokines, such as ovarian cancer and other forms of cancer. In some embodiments, provided herein are antibodies and other molecules that mediate increased T cell (e.g., CD 8)⁺T cells) activity or proliferation. Thus, in some embodiments, there is provided the use of such antibodies and other molecules in the treatment of diseases and disorders such as cancer that can be treated by increasing T cell activity or proliferation. In some embodiments, provided herein is the use of an antibody or other molecule described herein to mediate both increased T cell activity and increased T cell proliferation.

Upregulation of the immune system is particularly desirable in the treatment of cancer. In addition, BTN1a1 was specifically and highly expressed in cancer cells. The molecules described herein may also bind to cancer cells and cause their destruction by direct cytotoxicity or by ADCC or CDC mechanisms. Accordingly, provided herein are methods of treating cancer. Cancer refers to a tumor or tumor caused by abnormal uncontrolled cell growth. The cancer may be a primary cancer or a metastatic cancer.

In some embodiments, provided herein are methods of treating cancer by administering a molecule having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) that inhibits binding of BTN1a1 ligand to BTN1a 1. Cancers for which the treatment method may be useful include any malignant cell type, such as those found in solid tumors or hematologic cancers. Exemplary solid tumors include, but are not limited to, tumors of organs selected from the group consisting of pancreas, colon, caecum, esophagus, stomach, brain, head, neck, thyroid, thymus, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematologic cancers include, but are not limited to, bone marrow tumors, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. In some embodiments, the method further comprises administering an anti-PD 1 therapy or an anti-PD-L1 therapy.

In some embodiments, provided herein are methods of treating cancer in a subject by administering a molecule described herein that has an antigen-binding fragment that immunospecifically binds to BTN1a1 or a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule can inhibit binding of BTN1a1 ligand to BTN1a1, wherein the cancer can be breast cancer, neuroendocrine prostate cancer (NEPC), diffuse large B-cell lymphoma, melanoma, cancer from the national cancer institute panel (NCI 60), uveal melanoma, pancreatic cancer, ovarian cancer, uterine cancer, lung adenocarcinoma, desmoplastic small round cell tumor, bladder cancer, colorectal cancer, lung squamous cell carcinoma, liver cancer, lung cancer, stomach cancer, cholangiocellular carcinoma, squamous cell carcinoma, colon cancer, head and neck cancer, sarcoma, prostate cancer, liver cancer, pancreatic cancer, pheochromocytoma or paraganglioma (PCPG), cervical cancer, glioma, or Acute Myeloid Leukemia (AML). The molecule for treating cancer may be any molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule can inhibit binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) relative to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer). In some embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N55, N215, N449, or any combination thereof.

In some embodiments, provided herein are methods of treating cancer in a subject by administering a molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein the molecule can inhibit binding of BTN1a1 ligand to BTN1a1, wherein the cancer can be a squamous cell carcinoma of the lung, an adenocarcinoma of the prostate, an adenocarcinoma of the pancreas, or a hepatocellular carcinoma. The molecule for treating lung squamous cell carcinoma, prostate adenocarcinoma, pancreatic adenocarcinoma, or hepatocellular carcinoma can be any molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand, whereby the molecule inhibits the binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) relative to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer). In some embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N55, N215, N449, or any combination thereof.

In some embodiments, provided herein are methods of treating cancer in a subject by administering a molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), by which molecule binding of BTN1a1 ligand to BTN1a1 can be inhibited, the cancer being an anti-PD-1 therapy or an anti-PD-LL therapy resistant or refractory cancer. The molecule for use in treating an anti-PD-1 therapy or an anti-PD-L1 therapy resistant or refractory cancer is any molecule that can have an antigen-binding fragment that immunospecifically binds to a BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) as described herein, whereby the molecule inhibits binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) relative to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer). In some embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N55, N215, N449, or any combination thereof. In certain embodiments, the cancer is a cancer resistant or refractory to an anti-PD-1 therapy. In certain embodiments, the cancer is a cancer resistant or refractory to an anti-PD-L1 therapy. In certain embodiments, the anti-PD-1 therapy resistant or refractory cancer is a breast cancer or a lung cancer. In certain embodiments, the cancer resistant or refractory to an anti-PD-1 therapy is breast cancer or Lewis lung cancer. In certain embodiments, the cancer resistant or refractory to an anti-PD-1 therapy is breast cancer. In certain embodiments, the anti-PD-1 therapy resistant or refractory cancer is Lewis lung cancer.

Further examples of cancers that can be treated using the methods provided herein include, but are not limited to, malignancies, lymphomas, blastoma, sarcoma, leukemia, squamous cell cancer, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, mesothelioma), cancer of the peritoneum, hepatocellular cancer, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), esophageal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, melanoma, superficial melanoma, malignant macular melanoma, lentigo melanoma, and lentigo melanoma, Melanoma nodosa, uveal melanoma, germ cell tumors (yolk sac tumor, testicular cancer, malignant syncytial tumor), and B cell lymphomas (including low grade/follicular non-Hodgkin's lymphoma (NHL); Small Lymphocyte (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblast NHL; high grade lymphoblastic NHL; high grade small non-mitotic NHL; massive disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia), Chronic Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, multiple myeloma, Acute Myelogenous Leukemia (AML), and chronic myeloblastic leukemia.

The cancer may also be of any of the following histological types: malignant neoplasms; cancer; undifferentiated carcinoma; giant cell and spindle cell cancers; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphatic epithelial cancer; basal cell carcinoma; hair matrix cancer; metastatic cell carcinoma; papillary metastatic cell carcinoma; adenocarcinoma; malignant gastrinomas; cholangiocellular carcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma of familial colonic polyps; solid cancer; malignant carcinoid tumors; bronchoalveolar carcinoma; papillary adenocarcinoma; a cancer of the chromophobe; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic granulosa cancer; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; non-enveloped, hard-set cancers; adrenocortical carcinoma; endometrioid carcinoma; skin adjunct cancer; hyperhidrosis carcinoma; sebaceous gland cancer; succinic acid adenocarcinoma; mucoepidermoid carcinoma; cystic carcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory cancer; paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumors; malignant blastocyst cell tumors; malignant granulosa cell tumors; malignant osteoblastoma; podocyte carcinoma; malignant leydig cell tumor; malignant lipocytoma; malignant paraganglioma; malignant external paraganglioma of mammary gland; pheochromocytoma; vascular endothelioma; malignant melanoma; melanotic melanoma-free; superficial spreading melanoma; malignant melanoma in giant pigmented nevi; epithelial-like cell melanoma; malignant blue nevi; a sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; interstitial sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; a carcinosarcoma; malignant stromal tumors; malignant ovarian brenner's tumorigenicity; malignant phyllo-tumor; synovial sarcoma; malignant mesothelioma; clonal cell tumors; an embryonic carcinoma; malignant teratoma; malignant ovarian thyroid tumors; malignant syncytial tumor; malignant mesonephroma; vascular endothelioma; malignant vascular endothelioma; kaposi's sarcoma of the skin; malignant extravascular dermatoma; lymphangioleiomyosarcoma; osteosarcoma; near-cortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; malignant odontogenic tumors; amelogenic cell dental sarcoma; malignant ameloblastic tumors; an amelogenic fibrosarcoma; malignant pineal tumor; chordoma; malignant glioma; ependymoma; astrocytoma; primary plasma astrocytoma; fibroid astrocytoma; an astrocytoma; a glioblastoma; oligodendroglioma; oligodendroglioma; primary neuroectoderm; a sarcoma of the cerebellum; nodal cell neuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumors; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granulosa cell tumors; malignant lymphoma; hodgkin's disease; hodgkin's; granuloma-like; malignant lymphoma of small lymphocytes; large cell, diffuse malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specific non-hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cellular leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; and hairy cell leukemia.

In some embodiments, provided herein are methods of treating cancer in a subject by administering a molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or a BTN1A ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein the molecule is capable of inhibiting the binding of BTN1A ligand to BTN1a1, wherein the cancer is a lung cancer, a prostate cancer, a pancreatic cancer, an ovarian cancer, a liver cancer, a head and neck cancer, a breast cancer, or a stomach cancer. In some embodiments, provided herein are methods of treating cancer in a subject by administering a molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1A or a BTN1A ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule can inhibit binding of BTN1a1 ligand to BTN1a1, whereby the cancer can be a lung cancer. The lung cancer may be non-small cell lung cancer (NSCLC). The lung cancer may be Small Cell Lung Cancer (SCLC). The NSCLC may be squamous NSCLC. The molecule for treating lung cancer may be any molecule having an antigen binding fragment that immunospecifically binds BTN1a1 or glycosylated BTN1A as described herein. In some embodiments, the antigen binding fragment preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N55, N215, N449, or any combination thereof. In some embodiments, the method further comprises administering an anti-PD 1 therapy or an anti-PD-L1 therapy.

In some embodiments, provided herein are methods of treating cancer in a subject by administering a molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1A or a BTN1A ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein the molecule can inhibit binding of BTN1a1 ligand to BTN1a1, wherein the cancer can be a prostate cancer. The molecule for treating prostate cancer may be any molecule described herein having an antigen binding fragment that immunospecifically binds BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule can inhibit binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) relative to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer). In some embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N55, N215, N449, or any combination thereof. In some embodiments, the method further comprises administering an anti-PD 1 therapy or an anti-PD-L1 therapy.

In some embodiments, provided herein are methods of treating cancer in a subject by administering a molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein the molecule can inhibit binding of BTN1a1 ligand to BTN1a1, wherein the cancer can be pancreatic cancer. The molecule for treating pancreatic cancer may be any molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule can inhibit binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) relative to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer). In some embodiments, the method further comprises administering an anti-PD 1 therapy or an anti-PD-L1 therapy.

In some embodiments, provided herein are methods of treating cancer in a subject by administering a molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), wherein the molecule is capable of inhibiting the binding of BTN1a1 ligand to BTN1a1, whereby the cancer may be ovarian cancer. The molecule for treating ovarian cancer may be any molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule can inhibit binding of BTN1a1 ligand to BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) relative to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer). In some embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N55, N215, N449, or any combination thereof. In some embodiments, the method further comprises administering an anti-PD 1 therapy or an anti-PD-L1 therapy.

In some embodiments, provided herein are methods of treating cancer in a subject by administering a molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule inhibits the binding of BTN1a1 ligand to BTN1a1, whereby the cancer may be liver cancer. The molecule for treating liver cancer can be any molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). In some embodiments, the antigen binding fragment preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) relative to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer). In some embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N55, N215, N449, or any combination thereof. In some embodiments, the method further comprises administering an anti-PD 1 therapy or an anti-PD-L1 therapy

In some embodiments, provided herein are methods of treating a cancer in a subject by administering a molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule inhibits binding of BTN1a1 ligand to BTN1a1, whereby the cancer may be a head and neck cancer. The molecule for treating head and neck cancer can be any molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). In some embodiments, the antigen binding fragment preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) relative to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer). In some embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N55, N215, N449, or any combination thereof. In some embodiments, the method further comprises administering an anti-PD 1 therapy or an anti-PD-L1 therapy.

In some embodiments, provided herein are methods of treating cancer in a subject by administering a molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule inhibits binding of BTN1a1 ligand to BTN1a1, whereby the cancer may be breast cancer. The molecule for treating breast cancer can be any molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). In some embodiments, the antigen binding fragment preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) relative to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer). In some embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N55, N215, N449, or any combination thereof. In some embodiments, the method further comprises administering an anti-PD 1 therapy or an anti-PD-L1 therapy.

In some embodiments, provided herein are methods of treating cancer in a subject by administering a molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), whereby the molecule inhibits binding of BTN1a1 ligand to BTN1a1, whereby the cancer may be gastric cancer. The molecule for treating gastric cancer can be any molecule described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). In some embodiments, the antigen binding fragment preferentially binds glycosylated BTN1a1 relative to non-glycosylated BTN1a 1. In some embodiments, the antigen binding fragment preferentially binds to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) relative to BTN1a1 monomer (e.g., glycosylated BTN1a1 monomer). In some embodiments, the antigen-binding fragment immunospecifically masks BTN1a1 glycosylation at position N55, N215, N449, or any combination thereof. In some embodiments, the method further comprises administering an anti-PD 1 therapy or an anti-PD-L1 therapy.

5.4.2 methods of administration

Also provided herein are methods of administering a therapeutically effective amount of an antibody or molecule provided herein to a patient in need thereof using an anti-BTN 1a1 antibody, an anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) antibody, or other molecule having an antigen binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand, as an anti-tumor agent. In some embodiments, the patient is a cancer patient.

Various delivery systems are also known and may be used to administer the anti-BTN 1a1 anti-or other molecules with antigen-binding fragments that immunospecifically bind to BTN1a1, glycosylated BTN1a1, or BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer), or related pharmaceutical compositions, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or fusion protein, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, j. biol. chem.262: 4429-.

Methods of administration provided herein include, but are not limited to, injection, such as by parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In certain embodiments, the antibody, other molecule, or pharmaceutical composition provided herein is administered intramuscularly, intravenously, subcutaneously, intravenously, intraperitoneally, orally, intramuscularly, subcutaneously, intraluminal, transepidermally, or epicutaneously. The compositions may be administered by any convenient route, e.g., by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other bioactive agents. Administration may be systemic or local. In addition, pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosolized agent. See, for example, U.S. patent nos.6,019,968; 5,985, 20; 5,985,309, respectively; 5,934,272, respectively; 5,874,064, respectively; 5,855,913, respectively; 5,290,540, respectively; and 4,880,078; and PCT publication nos. wo 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903; all of which are incorporated herein by reference in their entirety. In certain embodiments, the antibodies, other molecules, or pharmaceutical compositions provided herein are administered locally to an area in need of treatment, which can be achieved by, for example, local infusion, by injection, or by means of an implant that is a porous, non-porous, or gelatinous material, including membranes, e.g., elastic membranes or fibers. In certain embodiments, when administering an antibody or other molecule described herein, care is taken to use a material that is not absorbed by the antibody or other molecule.

In certain embodiments, the humanized or chimeric antibodies provided herein are formulated in liposomes for targeted delivery. Liposomes are vesicles that encapsulate an aqueous phase, comprising concentric oriented phospholipid bilayers. Liposomes generally have various types of lipids, phospholipids, and/or surfactants. The components of liposomes are typically arranged in a bilayer structure, similar to the lipid profile of biological membranes. Liposomes may be useful delivery vehicles, in part due to their biocompatibility, low immunogenicity, and low toxicity. Methods of preparing liposomes are known in the art and are provided herein, see, e.g., pstein et al,1985, proc.natl.acad.sci.usa,82: 3688; hwang et al,1980Proc.Natl.Acad.Sci.USA,77: 4030-4; U.S. patent nos.4,485,045 and, 544,545; all of which are incorporated herein by reference in their entirety.

Also provided herein are methods of making liposomes having an extended serum half-life, i.e., enhanced circulation time, such as those disclosed in U.S. patent No.5,013,556. In certain embodiments, the liposomes used in the methods provided herein do not clear rapidly from the circulation, i.e., are not taken up by the Mononuclear Phagocyte System (MPS). Also provided herein are sterically (sterilly) stabilized liposomes prepared using common methods known to those skilled in the art. Sterically stabilized liposomes may contain lipid components with bulky and highly flexible hydrophilic moieties that reduce unwanted reactions of the liposome with serum proteins, reduce opsonization with serum components, and reduce recognition of MPS. Sterically stabilized liposomes can be prepared using polyethylene glycol. For the preparation of liposomes and sterically stabilized liposomes, see, e.g., Bendas et al,2001BioDrugs,15(4): 215-); allen et al,1987FEBSLett.223: 42-6; klibanov et al,1990FEBS Lett,268: 235-7; blum et al,1990, Biochim.Biophys.acta.,1029: 91-7; torchilin et al,1996, J.Liposome Res.6: 99-116; litzinger et al,1994, biochim. biophysis. acta,1190: 99-107; maruyama et al,1991, chem.pharm.Bull,39: 1620-2; klibanov et al,1991, Biochim Biophys Acta, 1062; 142-8 parts of; allen et al,1994, adv. drug Deliv. Rev,13: 285-.

Also provided herein are liposomes suitable for targeting to specific organs, see, e.g., U.S. patent No.4,544,545, or for targeting to specific cells, see, e.g., U.S. patent publication No.2005/0074403, which are incorporated herein by reference in their entirety. Liposomes particularly useful in the compositions and methods provided herein can be produced by reverse phase evaporation methods with lipid compositions comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through a filter material of defined pore size to produce liposomes of a desired diameter. In certain embodiments, molecules having antigen-binding fragments, such as F (ab') can be conjugated to liposomes using methods described earlier, e.g., Martin et al,1982, J.biol.chem.257:286-288, incorporated herein by reference in its entirety.

The humanized or chimeric antibodies described herein can also be formulated as immunoliposomes. Immunoliposomes refer to a liposome composition in which an antibody or fragment thereof is covalently or non-covalently attached to the surface of the liposome. The chemistry of attaching antibodies to liposome surfaces is known in the art, see, e.g., U.S. patent nos.6,787,153; allen et al,1995, Stealth Liposomes, Boca Rotan: CRC Press, 233-44; hansen et al,1995, Biochim. Biophys. acta,1239:133-144, which are incorporated herein by reference in their entirety. In certain embodiments, the immunoliposomes used in the methods and compositions provided herein are further sterically stabilized. In certain embodiments, the humanized antibodies described herein are covalently or non-covalently linked to a hydrophobic anchor stably rooted in the lipid bilayer of the liposome.Examples of hydrophobic anchors include, but are not limited to, phospholipids, e.g., Phosphatidylethanolamine (PE), Phosphatidylinositol (PI). To achieve covalent attachment between the antibody and the hydrophobic anchor, any biochemical strategy known in the art can be used, see, e.g., j.thomas August ed.,1997,Gene Therapy:Advances in

Pharmacology

volume 40, Academic Press, San Diego, Calif, p.399-435, incorporated herein by reference in their entirety. For example, a functional group on the antibody molecule can react with a reactive group on a hydrophobic anchor associated with the liposome, e.g., the amino group of a lysine side chain on the antibody can be coupled with liposome-associated N-glutaryl phosphatidylethanolamine activated with a water-soluble carbodiimide; or the thiol group of the reducing antibody may be coupled to the liposome via a thiol-reactive anchor, such as pyridylsulfonylpolylphosphatidylethanolamine. See, e.g., Dietrich et al,1996, Biochemistry,35: 1100-; loughrey et al,1987, Biochim.Biophys.acta,901: 157-160; martin et al,1982, J.biol.chem.257: 286-288; martin et al,1981, Biochemistry,20:4429-38, incorporated herein by reference in their entirety. Immunoliposomal formulations with an anti-BTN 1a1 antibody or other molecule with an antigen-binding fragment that immunospecifically binds BTN1a1 or glycosylated BTN1a1 are particularly useful as therapeutic agents because they deliver the active ingredient to the cytoplasm of the target cell (i.e., the cell that includes the receptor to which the antibody is bound). In certain embodiments, the immunoliposomes can have an increased half-life in the blood, specifically target cells, and can be internalized in the cytoplasm of the target cells, thereby avoiding loss or degradation of therapeutic agents of the endolysosomal pathway.

The immunoliposome compositions provided herein can have one or more vesicle-forming lipids, an antibody or other molecule of the present invention or a fragment or derivative thereof, and, optionally, a hydrophilic polymer. Vesicle-forming lipids can be lipids having two hydrocarbon chains, e.g., an acyl chain and a polar head group. Examples of vesicle-forming lipids include phospholipids, e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, neurophosphates and glycolipidsFor example, cerebrosides, gangliosides. Other lipids useful in the formulations provided herein are known to those skilled in the art and are encompassed in the present specification. In certain embodiments, the immunoliposome composition further comprises a hydrophilic polymer, for example, polyethylene glycol and the ganglioside GM1, which increases the serum half-life of the liposome. Methods of conjugating hydrophilic polymers to liposomes are well known in the art and are encompassed within the present specification. Other exemplary immunoliposomes and methods for preparing them may be found, for example, in U.S. patent application publication No. 2003/0044407; PCT International publication No. WO 97/38731, Vigerhoeads et al,1994, Immunomethods,4: 259-72; maruyama,2000, biol.pharm.Bull.23(7): 791-799; abra et al,2002, Journal of lipid Research,12 (1)&2):1-3；Park,2002,Bioscience Reports,22(2):267-281；Bendas et al,2001BioDrugs,14(4):215-224,J.Thomas August ed.,1997,Gene Therapy:Advances in

Pharmacology

Volume 40, Academic Press, San Diego, Calif, p.399-435; all of which are incorporated herein by reference in their entirety.

Also provided herein are methods of treating cancer patients by administering to the patient a unit dose of anti-BTN 1a1, anti-BTN 1a1 antibody, or other molecule having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). A unit dose refers to a physically discrete unit suitable as a unit dose of the subject, each unit containing a predetermined quantity of active material in association with a required diluent, i.e., carrier or vehicle, calculated to produce the desired therapeutic effect.

The antibody, molecule or composition is administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The amount to be administered depends on the subject to be treated, the ability of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. The precise amount of active ingredient to be administered depends on the judgment of the practitioner and is specific to each individual subject. However, suitable dosage ranges for systemic application are disclosed herein, depending on the route of administration. Suitable means of initiating and enhancing administration are also contemplated, and generally include an initial administration followed by repeated administrations at one or more hour intervals by subsequent injections or other administrations. Exemplary multiple administrations are described herein, useful for maintaining sustained high serum and tissue levels of the polypeptide or antibody. Alternatively, a continuous intravenous infusion sufficient to maintain the concentration in the blood within the range prescribed by the in vivo treatment is contemplated.

A therapeutically effective amount is a predetermined amount that is planned to achieve a desired effect. In general, the dosage will vary depending on the age, condition, sex and extent of the disease of the patient and can be determined by one skilled in the art. The dosage may be adjusted by an independent physician in the case of any complication.

In certain embodiments, the antibodies, molecules, or pharmaceutical compositions provided herein are packaged in a closed container, e.g., an ampoule or vial. In one embodiment, the antibodies, molecules, or pharmaceutical compositions provided herein are provided as a dry, sterile lyophilized powder or water-free concentrate in a closed container that can be reconstituted with, for example, water or saline to an appropriate concentration for administration to a subject. In certain embodiments, the antibodies, molecules or pharmaceutical compositions provided herein are provided as a dry sterile lyophilized powder in a closed container in a unit dose of at least 5mg, more preferably at least 10mg, at least 15mg, at least 25mg, at least 35mg, at least 45mg, at least 50mg or at least 75 mg. The lyophilized antibodies, molecules or pharmaceutical compositions provided herein should be stored in their original container between 2 ℃ and 8 ℃, and should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours or within 1 hour after reconstitution. In an alternative embodiment, the antibodies, molecules or pharmaceutical compositions provided herein are provided in liquid form in a closed container indicating the quantity and concentration of the antibodies, molecules or pharmaceutical compositions. In certain embodiments, the liquid form of the antibody, molecule or pharmaceutical composition provided herein is provided in a closed container at least 1mg/ml, more preferably at least 2.5mg/ml, at least 5mg/ml, at least 8mg/ml, at least 10mg/ml, at least 15mg/kg, at least 25mg/ml, at least 50mg/ml, at least 100mg/ml, at least 150mg/ml, at least 200 mg/ml.

The exact dose employed in the formulation will also depend on the route of administration, the severity of the condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. For anti-BTN 1a1 antibodies or other molecules having an antigen-binding fragment that immunospecifically binds to BTN1a1, glycosylated BTN1a1, or BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer), the dose administered to a patient is typically 0.01mg/kg to 100mg/kg of patient body weight. In certain embodiments, the dose administered to the patient is between 0.01mg/kg to 20mg/kg, 0.01mg/kg to 10mg/kg, 0.01mg/kg to 5mg/kg, 0.01 to 2mg/kg, 0.01 to 1mg/kg, 0.01mg/kg to 0.75mg/kg, 0.01mg/kg to 0.5mg/kg, 0.01mg/kg to 0.25mg/kg, 0.01 to 0.15mg/kg, 0.01 to 0.10mg/kg, 0.01 to 0.05mg/kg, or 0.01 to 0.025mg/kg of the patient's body weight. In particular, the dose administered to the patient may be 0.2mg/kg, 0.3mg/kg, 1mg/kg, 3mg/kg, 6mg/kg or 10 mg/kg. Doses as low as 0.01mg/kg are expected to show significant efficacy. Dosage levels of 0.10-1mg/kg are expected to be most suitable. Higher doses (e.g., 1-30mg/kg) are also expected to be active. Generally, human antibodies have a longer half-life in humans than antibodies from other species due to an immune response to the foreign polypeptide. Thus, lower doses of human antibodies and less frequent administration may be practical. Further, the dosage and frequency of administration of the antibodies or other molecules provided herein can be reduced by modifications such as lipidation to enhance uptake of the antibodies and tissue penetration.

In yet another embodiment, the composition may be delivered in a controlled or sustained release system. Any technique known to those skilled in the art can be used to produce sustained release formulations having one or more of the antibodies, molecules, or pharmaceutical compositions provided herein. See, for example, U.S. Pat. Nos.4,526,938; PCT publications WO 91/05548; PCT publications WO 96/20698; ning et al, radiothergy & Oncology 39: 179. minus 189(1996), Song et al, PDAjournal of Pharmaceutical Science & Technology 50: 372. minus 397 (1995); cleek et al, Pro.Int' l.Symp.control.Rel.Bioact.Mater.24:853-854 (1997); and Lam et al, Proc. int' l. Symp. control Rel. Bioact. Mater.24:759-760 (1997); all of which are incorporated herein by reference in their entirety. In one embodiment, the pump may be used in a controlled release system (see Langer, supra; Sefton,1987, CRC Crit. RefBiomed. Eng.14: 20; Buchwald et al,1980, Surgery88: 507; and Saudek et al,1989, iV. Engl. J. Med.321: 574). In another embodiment, polymeric materials may be used to achieve Controlled release of antibodies or polypeptides (see, e.g., Medical Applications of controlledRelease, Langer and Wise, eds.), CRC Pres, Boca Raton, Fla (1974); Controlled Drug bioavailability, Drug Product Design and Performance, Smolen and Ball, Wiley, New York (1984); Ranger and Peppas,1983, J., Macromol. Sci. Rev. Macromol Chem.23: 61; see, e al Levy et al,1985, Science 228: 190; During et al,1989, Ann. Neurol.25: 351; Howard et al,1989, J. Neuros.71: 105); U.S. patent nos.5,679,377; U.S. patent nos.5,916,597; U.S. patent nos.5,912,015; U.S. patent nos.5,989,463; U.S. patent nos.5,128,326; PCT publication nos. wo 99/15154; and PCT publication No. wo 99/20253); all of which are incorporated herein by reference in their entirety.

Examples of polymers that may be used in sustained release formulations include, but are not limited to, poly (-hydroxy ethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-vinyl acetate), poly (methacrylic acid), Polyglycolide (PLG), polyanhydrides, poly (N-vinyl pyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), Polylactide (PLA), poly (lactide-co-glycolide) (PLGA), and polyorthoesters. In yet another embodiment, the Controlled Release system may be placed in close proximity to the target of treatment (e.g., the lung), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in medical Applications of Controlled Release, supra, vol.2, pp.115-138 (1984)). In another embodiment, a polymeric composition useful as a controlled release implant according to Dunn et al (see U.S. patent No.5,945,155) is used, which is incorporated herein by reference in its entirety. Implantation can generally be performed anywhere within the body of a patient in need of treatment based on the therapeutic effect of in situ controlled release of bioactive material from a polymer system.

In another embodiment, a non-polymeric sustained release system is used, wherein a non-polymeric implant in the body of the subject is used as the drug delivery system. Upon implantation in the body, the organic solvent of the implant will dissipate, disperse or leach from the composition into the surrounding tissue fluids, and the non-polymeric material will gradually coagulate or precipitate to form a solid, microporous matrix (see U.S. patent No.5,888,533). Other controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-. Any technique known to those skilled in the art can be used to produce sustained release formulations comprising one or more therapeutic agents provided herein. See, for example, U.S. Pat. Nos.4,526,938; international publication nos. WO 91/05548 and WO 96/20698; ning et al,1996, radiothergy & Oncology 39: 179-189; song et al,1995, PDA Journal of Pharmaceutical Science & Technology 50: 372-; cleek et al,1997, Pro.int' l.Symp.control.Rel.Bioact.Mater.24: 853-854; and Lam et al,1997, Proc. int' l.Symp. control Re I.Bioact. Mater.24: 759-760; all of which are incorporated herein by reference in their entirety.

Also provided herein are embodiments wherein the composition has a nucleic acid encoding an antibody or other molecule provided herein, wherein the nucleic acid can be administered in vivo to facilitate expression of the antibody or polypeptide it encodes, by constructing it as part of a suitable nucleic acid expression vector, and administering it such that it becomes intracellular, e.g., by using a retroviral vector (see U.S. Pat. No.4,980,286), or by direct injection, or by using microprojectile bombardment (e.g., gene gun; Biolistic, Dupont), or covered with lipid or cell surface receptors or transfection reagents, by ligation with a homeobox-like peptide known to enter the nucleus (see, e.g., Joliot et al,1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868). Alternatively, the nucleic acid may be introduced intracellularly by homologous recombination and incorporated into the host cell DNA for expression.

In addition, treatment of a subject with a therapeutically effective amount of an antibody, other molecule, or pharmaceutical composition provided herein can include a single treatment or a series of treatments. It is contemplated that the antibodies, molecules, or pharmaceutical compositions provided herein can be administered systemically or locally to treat a disease, e.g., inhibit tumor cell growth or kill cancer cells in a cancer patient with locally progressing or metastatic cancer. They may be administered intravenously, intrathecally and/or intraperitoneally. They may be administered alone or in combination with antiproliferative agents. In one embodiment, they are administered to reduce the cancer burden of the patient prior to surgery or other procedure. Alternatively, they may be administered after surgery to ensure that any residual cancer (e.g., cancer that has not been removed by surgery) does not survive. In certain embodiments, they may be administered after remission of the primary cancer to prevent metastasis.

5.6 combination therapy

Also provided herein are compositions and methods comprising administering to a subject in need thereof an anti-BTN 1a1 antibody, an anti-BTN 1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) antibody, or other molecule having an antigen binding fragment that immunospecifically binds BTN1a1 or BTN1a1 ligand, in combination with a second therapy. In some embodiments, the subject is a cancer patient and the second therapy is an anti-cancer therapy or an anti-hyperproliferative therapy.

In certain embodiments, compositions and methods comprising administering the antibodies or other molecules provided herein, when used in combination with another anti-cancer therapy or anti-hyper-proliferative therapy, can enhance the therapeutic effect of the other anti-cancer therapy or anti-hyper-proliferative therapy. Thus, the methods and compositions described herein can be provided in combination with a second therapy to achieve a desired effect, e.g., killing cancer cells, inhibiting hyperproliferation of cells, and/or inhibiting cancer metastasis.

In certain embodiments, the second therapy has a direct cytotoxic effect, e.g., chemotherapy, targeted therapy, cryotherapy, hyperthermia, photodynamic therapy, High Intensity Focused Ultrasound (HIFU) therapy, radiation therapy, or surgical therapy. The targeted therapy may be a biological targeted therapy or a small molecule targeted therapy. In other embodiments, the second therapy has no direct cytotoxic effect. For example, the second therapy may be an agent that upregulates the immune system without a direct cytotoxic effect.

The second therapy may be an anti-PD 1 therapy or an anti-PD-L1 therapy.

Thus, in another aspect, the invention provides a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a molecule and a therapeutically effective amount of an anti-PD-1 therapy and/or an anti-PD-L1 therapy, wherein the molecule comprises an antigen-binding fragment that immunospecifically binds to BTN1a 1.

In certain embodiments, the method comprises administering an anti-PD-1 therapy. In certain embodiments, the method comprises administering an anti-PD-L1 therapy. In certain embodiments, the methods comprise administering an anti-PD-1 therapy and an anti-PD-L1 therapy.

In certain embodiments, the anti-PD-1 therapy or the anti-PD-L1 therapy comprises an anti-PD-1 or anti-PD-L1 antibody or antibody fragment, or a soluble PD-1 or PD-L1 ligand, or an Fc fusion protein thereof.

In some embodiments, the anti-PD-1 therapy comprises nivolumab (Opdivo), pembrolizumab (Keytruda), pidilizumab, AMP-514, AMP-224, or a combination thereof.

In some embodiments, the anti-PD-1 therapy comprises an anti-PD-1 antibody provided in international application PCT/US 20126/64394.

In some embodiments, the anti-PD-L1 therapy is yw243.55.s70, MPDL3280A, MEDI-4736, MSB-0010718C, MDX-1105, or a combination thereof.

In some embodiments, the anti-PD-L1 therapy includes antibodies provided in international application No. pct/US20126/024691, disclosed as WO2016/160792a1, and international application No. pct/US 2017/024027.

In some embodiments, the molecule comprising an antigen-binding fragment that immunospecifically binds BTN1a1 is formulated with an anti-PD-1 therapy and/or an anti-PD-L1 therapy.

In some embodiments, the molecule comprising an antigen-binding fragment that immunospecifically binds BTN1a1 is formulated separately from an anti-PD-1 therapy and/or an anti-PD-L1 therapy.

In some embodiments, the molecule comprising an antigen-binding fragment that immunospecifically binds BTN1a1 is administered independently at the same time or separately within a time interval as the anti-PD-1 therapy or anti-PD-L1 therapy, optionally followed by one or more repeat dosing cycles.

In some embodiments, the treatment produces at least one therapeutic effect selected from the group consisting of a reduction in tumor size, a reduction in the number of metastatic lesions over time, a complete response, a partial response, and a stable condition.

Methods provided herein include administering to a subject in need thereof an anti-BTN 1a1 antibody, an anti-BTN 1a1 ligand antibody, or other molecule having an antigen-binding fragment that immunospecifically binds to a BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in combination with a second or additional therapy (e.g., an anti-PD-1 therapy or an anti-PD-L1 therapy). The antibodies, other molecules, or pharmaceutical compositions provided herein can be administered before, during, after, or in various combinations relative to the second anti-cancer therapy. The interval of administration may be from simultaneous to minutes to days or weeks. In embodiments in which the antibody or other molecule described herein and the anti-cancer agent are provided to the patient separately, it is generally ensured that no significant time period is exceeded between the times of each delivery, such that the two compounds are still able to exert a beneficial combined effect on the patient. In such cases, it is contemplated that the antibody or other molecule provided herein and the second anticancer therapy can be provided to the patient within about 12 to 24 or 72 hours of each other, more particularly, within 6-12 hours of each other. In certain instances, the time period of treatment can be significantly extended, with days (2, 3, 4,5, 6, or 7 days) to weeks (1, 2, 3, 4,5, 6,7, or 8 weeks) elapsing between each administration.

In certain embodiments, the course of treatment will last from 1 to 90 days or more (this range includes the number of days in the interval). It is contemplated that one agent may be administered on any one or any combination of days 1 through 90 (this range includes alternate days), and that another agent may be administered on any one or any combination of days 1 through 90 (this range includes alternate days). Furthermore, after a course of treatment, it is desirable to have a period of time during which no anti-cancer therapy is administered. This time period may last from 1 to 7 days, and/or from 1 to 5 weeks, and/or from 1 to 12 months or more (this range includes the number of days in the interval), depending on the patient's condition, e.g., their prognosis, strength, health, etc. The treatment cycle may be repeated as necessary.

Various combinations may be employed. Listed below are some examples where the anti-BTN 1a1 antibody or other molecule described herein is "a" and the second anti-cancer therapy (e.g., anti-PD-1 therapy or anti-PD-L1 therapy) is "B": A/B/A B/A/B B/B/AA/A/B A/B/B B/A/A A/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/A B/B/A/A B/A/B/A B/A/B A/A/A/B B/A/A/A A/B/A A/A/B/A/A/A/B A/B/A/B A/B/B/B/A

Administration of any of the antibodies, molecules, or pharmaceutical compositions provided herein to a patient in combination with a second therapy (e.g., an anti-PD-1 therapy or an anti-PD-L1 therapy) will follow the general protocol for administering such a second therapy, if any, taking into account the toxicity of the second therapy. Thus, in certain embodiments, there is a step of monitoring toxicity attributable to the combination therapy.

Chemotherapy

A wide variety of chemotherapeutic agents may be used as the second therapy according to the current embodiments. The chemotherapeutic agent may be a compound or composition administered in the treatment of cancer. These agents or drugs can be classified by the way of their activity within the cell, e.g., whether and at what stage the cell cycle is affected. Alternatively, an agent can be characterized by its ability to directly cross-link DNA, to insert DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, for example, benzotepa (benzodopa), carbaquinone, meturedpa (meturedpa) and uredepa (uredpa); ethyleneimine and methylethyleneimine, including altretamine, triethylenemelamine (triethyleneimine), triethylenephosphoramide (triethylenephosphoramide), triethylenethiophosphoramide (triethylenethiophosphamide), and trimetylomelamine (trimetylomelamine); annonaceous acetogenins (in particular bullatacin and bullatacin); camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its Aldocosan, Kazelesin and Bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 2); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, for example chlorambucil, chlorophosphamide, estramustine, ifosfamide, mechlorethamine oxide hydrochloride, melphalan, neomustard (novembichin), cholesterol chlorambucil, prednimustine, trofosfamide and uracil mustard; nitrosoureas (nitrosurea), e.g., carmustine, pyritinose, fotemustine, lomustine, nimustine, and ranimustine (ranimustine); antibiotics, for example, enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma and calicheamicin omega); daptomycin (dynemicin), including daptomycin a; bisphosphonates, for example, clodronate (clodronate); esperamicin (esperamicin); and neocarzinostain chromophores and related chromoproteins enediyne antimetabolites chromophores), aclacinomycin (aclacinomycin), actinomycin (actinomycin), anthranomycin (aurramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), carubicin (carabicin), carminomycin (caminomycin), carcinomycin (carzinophilin), chromomycin (chromomycin), dactinomycin (dactinomycin), daunomycin, ditorexin (detroribin), 6-diazo-5-side oxy-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin and deoxydoxorubicin), epirubicin (epirubicin), mitorubicin (idarubicin), idarubicin (idarubicin), doxorubicin (mitomycin), mitomycin (mitomycin), clarithromycin (azamycin C), clarithromycin (e.g., Mycophenolic acid (mycophenolic acid), nogomycin (nogalamycin), olivomycin (olivomycin), pelomycin (pelomomycin), profomycin (popryomycin), puromycin (puromycin), quinamycin (quelamycin), roxobicin (rodorubicin), streptomycin (streptonigrin), streptozocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), zinostatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin (denopterin), pteropterin (pteropterin), trimetrexate (trimetrexate); purine analogs, such as fludarabine (fludarabine), 6-mercaptopurine, thiamiazines, thioguanine; pyrimidine analogs, for example, cyclocytidine, azacitidine, 6-azauridine, carmofur (carmofur), cytarabine, dideoxyuridine, deoxyfluorouridine, enocitabine (enocitabine), fluorouridine; androgens such as caridotestosterone (calusterone), dromostanoloneproprione, epitioandrostanol (epitiostanol), mepiquane (mepiquitane), testolactone (testolactone); anti-adrenal agents, e.g., mitotane, trilostane (trilostane); folic acid replenisher, e.g., leucovorin (Frolinic acid); acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bessburyl (beslabucil); a bisantrene group; edatrexate (edatraxate); desphosphamide (defofamine); colchicine (demecolcine); diazaquinone (diaziqutone); ilonidine (elfosmithine); ammonium etiolate (ellitiniumacetate); epothilone (epothilone); ethydine (etoglucid); gallium nitrate; a hydroxyurea; mushroom polysaccharides (lentinan); lonidamine (lonidainine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone; mopidanol (mopidanmol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllic acid); 2-acethydrazide; procarbazine; PSK polysaccharide complex; razoxane (rizoxane); rhizomycin (rhizoxin); azofurans (sizofurans); germanium spiroamines (spirogyranium); tenuizonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2', 2-trichlorotriethylamine; trichothecenes (trichothecenes) (particularly T-2 toxin, myxomycin A (veracurin A), bacillocin A (roridin A) and serpentin (anguidine)); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannomustine (manomostine); dibromomannitol; dibromodulcitol (mitolactol); pipobromane (pipobroman); gatifloxacin (gacytosine); cytarabine ("Ara-C"); cyclophosphamide; taxoids, such as paclitaxel and docetaxel gemcitabine (docetaxel) 6-thioguanine; mercaptopurine; platinum coordination complexes, e.g., cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (vinorelbine); norfloxacin (novantrone); teniposide (teniposide); edatrexae; daunorubicin; aminopterin; (xiloda); ibandronate (ibandronate); irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, for example, retinoic acid; capecitabine (capecitabine); carboplatin, procarbazine, Lincomycin (Lincomycin), gemcitabine, navelbine, farnesyl protein transferase inhibitors, antiplatin; and a pharmaceutically acceptable salt, acid or derivative of any of the above.

Radiotherapy

Other conventional anticancer therapies that can be used in combination with the methods and compositions described herein are radiation therapy, or radiation therapy. Radiation therapy involves the use of gamma-rays, X-rays, and/or the direct delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam radiation (U.S. Pat. Nos.5,760,395 and 4,870,287; all of which are incorporated herein by reference in their entirety), and UV-irradiation. Most likely all of these factors exert extensive damage to DNA, to the precursors of DNA, to the replication and repair of DNA, and to the assembly and maintenance of chromosomes.

The tumor microenvironment is inherently suppressive due to the presence of myeloid-derived suppressor and regulatory T cells, which infiltrate the tumor and act to suppress immune responses. Furthermore, expression of certain inhibitory molecules on T cells and Antigen Presenting Cells (APCs) may limit the effective immune response. Radiation mediates anti-tumor effects by inducing apoptosis, senescence, autophagy of tumor cells, and in some cases, may stimulate a more effective immune response.

Radiation can be a means of placing tumor cells under stress conditions, enabling the tumor cells to activate mechanisms that resist stress. Molecules activated under such stress conditions can be used as targets for therapy in combination with radiation. BTN1a1 was identified as a potential target for overexpression under such conditions.

Molecules having antigen-binding fragments that immunospecifically bind to BTN1a1 or BTN1a1 ligands (e.g., GAL-1, GAL-9, NRP-2, BTLA) described herein can stimulate local and systemic immune responses. In some embodiments, a therapeutically effective amount of an antibody, other molecule, or pharmaceutical composition described herein is administered prior to, concurrently with, or after radiation therapy to achieve a synergistic effect.

In some embodiments, a therapeutically effective amount of an antibody, other molecule, or pharmaceutical composition described herein is administered that is effective to sensitize a tumor in a host to radiation. The radiation may be ionizing radiation, in particular gamma radiation. In some embodiments, the gamma radiation is emitted by a linear accelerator or by a radionuclide. The irradiation of the tumor by the radionuclide may be external or internal.

In some embodiments, administration of the antibodies, other molecules or pharmaceutical compositions described herein is initiated up to a month, particularly 10 days or a week, prior to tumor irradiation. In addition, the irradiated tumors are divided and administration of the antibodies, other molecules, or pharmaceutical compositions described herein is maintained in the interval between the first and last irradiation periods.

The radiation may also be X-ray radiation, gamma-ray radiation, or charged particle radiation (proton beam, carbon beam, helium beam) (or "radiation" in general). The radiation dose ranges from 50 to 600X-ray doses per day to 800 to 6000X-ray single doses over certain intervals (2 or more days to weeks). The radiation may be administered once daily, twice daily, three times daily or four times daily. The dosage range of radioisotopes varies widely, and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake by tumor cells.

Targeted therapy

Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") involved in the growth, development and spread of cancer. Targeted cancer therapies are also referred to as "molecular targeted drugs," "molecular targeted therapies," "precision drugs," or similar names. Unlike standard chemotherapy, which generally acts on all rapidly dividing normal and cancer cells, targeted therapies act on specific molecular targets associated with cancer.

Targeted therapies include small molecule targeted therapies and biological targeted therapies, e.g., monoclonal antibodies. Small molecule compounds are generally developed to target sites located inside cells because these agents are able to enter cells relatively easily. Biological targeted therapies such as monoclonal antibodies are often used for targets outside or on the cell surface.

Many different targeted therapies have been approved for cancer treatment. These therapies include hormone therapy, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, immunotherapy, and toxin delivery molecules.

Hormone therapy slows or stops the growth of hormone sensitive tumors, which require certain hormones for their growth. Hormone therapy works by preventing the body from producing the hormone, or by interfering with the action of the hormone. Hormone therapy has been approved for breast and prostate cancer.

Signal transduction inhibitors block the activity of molecules involved in signal transduction by which a cell responds to signals from its environment. During this process, once a cell receives a particular signal, the signal is relayed within the cell through a series of biochemical reactions, ultimately producing an appropriate response. In some cancers, malignant cells are stimulated to divide continuously, rather than being promoted to do so by external growth factors. Signal transduction inhibitors interfere with this inappropriate signal.

Gene expression regulators modify the function of proteins that play a role in regulating gene expression. Inducers of apoptosis cause cancer cells to undergo a process that controls cell death, known as apoptosis. Apoptosis is a method used by the body to remove unwanted or abnormal cells, but cancer cells possess strategies to avoid apoptosis. Inducers of apoptosis may circumvent these strategies to cause cancer cell death.

Angiogenesis inhibitors block new blood vessel growth to tumors (a process known as tumor angiogenesis). Blood supply is necessary for tumor growth beyond a certain size because blood provides the oxygen and nutrients needed for tumor growth to continue. Treatments that interfere with angiogenesis can block tumor growth. Certain targeted therapies that inhibit angiogenesis interfere with the activity of Vascular Endothelial Growth Factor (VEGF), a substance that stimulates neovascularization. Other angiogenesis inhibitors target other molecules that stimulate new blood vessel growth.

Immunotherapy triggers the immune system to destroy cancer cells. Certain immunotherapies are monoclonal antibodies, which recognize specific molecules on the surface of cancer cells. Binding of the monoclonal antibody to the target molecule causes immune destruction of the cell expressing the target molecule. Other monoclonal antibodies bind to certain immune cells to help these cells better kill cancer cells.

Monoclonal antibodies that deliver toxic molecules can specifically cause the death of cancer cells. Once the antibody binds to its target cell, a toxic molecule, such as a radioactive substance or toxic chemical, attached to the antibody is accepted by the cell, eventually killing the cell. The toxin will not affect the cells that lack the target of the antibody, i.e., the vast majority of cells in the body.

Cancer vaccines and gene therapy are also considered targeted therapies because they interfere with the growth of specific cancer cells.

For example, the following provides a list of FDA-approved targeted therapies that can be used as the second therapy according to the present embodiments.

Adenocarcinoma of the stomach or gastroesophageal junction: trastuzumab

Ramurumumab (ramucirumab)

Basal cell carcinoma: vismodegib (vismodegib) (Erivedge)^TM) Sonidergib (sonidegib)

Brain cancer: bevacizumab

Everolimus (everolimus)

Breast cancer: everolimus

Tamoxifen, toremifene

Trastuzumab

Fulvestrant

Anastrozole

Exemestane

Lapatinib

Letrozole

Pertuzumab (pertuzumab)

Addo-trastuzumab emtansine (ado-trastuzumab emtansine)

Pabociclib

Cervical cancer: bevacizumab

Colorectal cancer: cetuximab

Panitumumab

Bevacizumab

Abiracy (ziv-aflibercept)

Ruighfenib (regorafenib)

Ramoplurumab

Dermatofibrosarcoma protruberance: imatinib mesylate

Endocrine/neuroendocrine tumors: lanreotide acetate (Lanreotide acetate)

Head and neck cancer: cetuximab

Gastrointestinal stromal tumors: imatinib mesylate

Sunitinib (sunitinib)

Regorafenib

Giant cell tumors of the skeleton: dinoteumab (denosumab)

Kaposi sarcoma: aliretin A acid (alitretinin)

Renal cancer: bevacizumab

Sorafenib

Sunitinib

Pazopanib

Temsirolimus (temsirolimus)

Everolimus

Cetinib

Leukemia: retinoic acid

Imatinib mesylate

Dasatinib

Nilotinib

Bosutinib

Rituximab

Alemtuzumab (alemtuzumab)

Oxamumumab (ofatumumab)

Orabine eutuzumab (obinutuzumab)

Ibrutinib (ibrutinib) (Imbruvica)^TM) Edarani (idelalisib)

Bonatuzumab (Blincyto)^TM)

Liver cancer: sorafenib

Lung cancer: bevacizumab

Crizotinib (crizotinib)

Erlotinib

Gefitinib

Afatinib dimaleate

Ceritinib (LDK378/Zykadia), ramucirumab

Nivolumab

Pembrolizumab

Lymphoma: ibritumomab tiuxetan

Deni (r) -Ni)Interleukin (denileukin difitox)

Bentuximab (brentuximab vedotin)

Rituximab

Vorinostat (vorinostat)

Romidepsin (romidepsin)

Bexarotene (bexarotee)

Bortezomib

Pralatrexate

Lenalidomide

Ibrutinib (Imbruvica)^TM) Stituximab (siltuximab) (Sylvant)^TM) Edarani, Adelab

Belinostat (Beleodaq)^TM)

Melanoma: ipilimumab

Vemurafenib

Trametinib

Dalafini (dabrafenib)

Pembrolizumab

Nivolumab

Multiple myeloma: bortezomib

Carfilzomib (carfilzomib)

Lenalidomide

Pomalidomide

panobinostat

Myelodysplastic/myeloproliferative disorders: imatinib mesylate

Lukuconi (ruxolitinib) phosphate (Jakafi)^TM)

Neuroblastoma: datuximab (dintuximab) (Unituxin)^TM)

Ovarian epithelial/fallopian tube/cloudy-eruptive peritoneal carcinoma: bevacizumab

Olaparib (Lynparza)^TM)

Pancreatic cancer: erlotinib

Everolimus

Sunitinib

Prostate cancer: cabazitaxel (cabazitaxel)

Enzalutamide (enzalutamide)

Abiraterone acetate

Radium 223 chloride

Soft tissue sarcoma: pazopanib

Systemic mastocytosis: imatinib mesylate

Thyroid cancer: cabotinib (Cometriq)^TM) Vandetanib

Sorafenib

Levatinib mesylate (Lenvima)^TM)

Immunotherapy

The skilled artisan will appreciate that immunizationThe therapies can be used in combination or together with the methods of the embodiments. In the context of cancer therapy, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab

Is an example of this. Checkpoint inhibitors, e.g., ipilimumab, are another such example. The immune effector can be, for example, an antibody specific for certain markers on the surface of tumor cells. The antibody alone may act as an effector of therapy, or it may recruit other cells to actually affect cell killing. The antibody may also be conjugated to a drug or toxin (e.g., chemotherapeutic agent, radionuclide, ricin a chain, cholera toxin, pertussis toxin), merely serving as a targeting agent. Alternatively, the effector may be a lymphocyte with a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

In certain embodiments, the immunotherapy is an anti-PD-1 therapy or an anti-PD-L1 therapy.

anti-PD-1 therapy may include any inhibitor of PD-1. In certain embodiments, the anti-PD-1 therapy can include an anti-PD-1 antibody or antigen-binding fragment thereof, an inhibitory nucleic acid, or a soluble PD-1 ligand (e.g., soluble PD-L1), or a fusion protein thereof (e.g., Fc-fusion protein). In certain embodiments, the anti-PD-1 therapy comprises nivolumab (Opdivo), pembrolizumab (Keytruda), pidilizumab, AMP-514, or AMP-224.

In some embodiments, the anti-PD-1 therapy comprises nivolumab (CAS registry number 946414-94-4). Nivolumab is also known as MDX-1106, MDX-1106-04, ONO-4538 or BMS-936558. Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in US8,008,449 and WO 2002/121168.

In some embodiments, the anti-PD-1 therapy comprises pembrolizumab. Pembrolizumab also known as

Lammbrolizumab, Merck3745, MK-3475 or SCH-900475. Pembrolizumab is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab is disclosed in, for example, Hamid, O et al (1973) New England journal Medicine 369(2):134-44, WO2002/114335, and US 8354509.

In some embodiments, the anti-PD-1 therapy is pidilizumab. Pidizumab, also known as CT-011(CureTech), is a humanized IgG1 monoclonal antibody that binds to PD-1. Pidizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO 2009/101611.

In some embodiments, the anti-PD-1 therapy comprises an anti-PD-1 antibody provided in International application PCT/US 2016/64394.

Other anti-PD 1 antibodies useful as anti-PD 1 therapies are disclosed in US8,609,089, US2010028330 and/or US 20120114649.

In some embodiments, the anti-PD-1 therapy includes the fusion protein AMP514 (amplimune). AMP-224, also known as B7-DCIg, is disclosed, for example, in WO2010/027827 and WO 2011/066342. AMP-224 is a PD-L2Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1.

In certain embodiments, the anti-PD-1 therapy comprises an immunoadhesin (e.g., an immunoadhesin comprising an extracellular portion of PD-L1 or PD-L2 or a PD-1 binding portion fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In certain embodiments, the anti-PD-1 therapy comprises the fusion protein AMP-224 (Fc fusion of PD-L2).

anti-PD-L1 therapy may include any inhibitor of PD-L1. In certain embodiments, the anti-PD-1 therapy can include anti-PD-L1 or an antigen-binding fragment thereof, an inhibitory nucleic acid, or a soluble PD-L1 ligand (e.g., soluble PD-1), or a fusion protein thereof (e.g., Fc-fusion protein). In certain embodiments, the anti-PD-L1 therapy comprises yw243.55.s70, MPD13280A, MEDI-4736, MSB-0010718C, or MDX-1105.

In some embodiments, the anti-PD-L1 therapy comprises MDX-1105. MDX-1105 is also known as BMS-936559. See, for example, WO 2002/005874.

In some embodiments, the PD-LL therapy comprises antibody yw243.55.s70, as described, for example, in WO2010/077634 (heavy and light chain variable region sequences are shown in SEQ ID NOs 20 and 21, respectively).

In some embodiments, the PD-L1 therapy comprises MDPL3280A (Genentech/Roche). MDPL3280A is a human Fc optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies against PD-L1 are disclosed, for example, in U.S. Pat. No.7943743 and U.S. publication No. 20120039906.

In some embodiments, the anti-PD-L1 therapy comprises antibody MSB0010718C (Merck Serono). MSB0010718C is also known as A09-246-2.

In some embodiments, the anti-PD-L1 therapy comprises MDPL3280A (Genentech/Roche), a human Fc-optimized IgG1 monoclonal antibody that binds PD-L1. MDPL3280A and other human monoclonal antibodies against PD-L1 are disclosed in U.S. Pat. No.7943743 and U.S. publication No. 20120039906.

In some embodiments, the anti-PD-L1 therapy includes antibodies provided in international application No. pct/US20126/024691, publication nos. WO2016/160792 and international application No. pct/US 2017/024027.

In one aspect of immunotherapy, tumor cells carry certain markers that can be targeted, i.e., not present on most other cells. There are a number of tumor markers, any of which may be suitable for targeting in the context of the current embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and pl 55. An alternative aspect of immunotherapy is the combination of anti-cancer and immunostimulatory effects. Immunostimulatory molecules also exist, including: cytokines, e.g., IL-2, IL-4, IL-12, GM-CSF, γ -IFN, chemokines, e.g., MIP-1, MCP-1, IL-8, and growth factors, e.g., FLT3 ligand.

Examples of immunotherapies currently under investigation or in use are immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Pat. Nos.5,801,005 and 5,739,169; Hui and Hashimoto, Infect Immun, 66(11):5329-36 (1998); Christodoulides et al, Microbiology,66(11):5329-36 (1998); cytokine therapies such as interferons α, β and γ, IL-1, GM-CSF and TNF (Bukowski et al, Clin Cancer 865, 4(10):2337-47 (1998); Davidson et al, J2012 thermal, 21(5):389-98(1998), Heltrals et, Acicola 37-347, 1998), and anti-rat-5 antibodies such as anti-rat-5, and mouse, as anti-9, and mouse, as anti-2, and mouse, as anti-2.

Surgery

Approximately 60% of cancer patients will undergo some type of surgery, including preventative, diagnostic or staging, curative and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, resected, and/or destroyed, and may be used in conjunction with other therapies, such as the treatment of the present embodiment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to the physical removal of at least a portion of a tumor. Surgical treatments, other than tumor resection, include laser surgery, cryosurgery, electrosurgery, and microsurgery (morse).

Upon resection of some or all of the cancerous cells, tissue, or tumor, a cavity may form in the body. The treatment may be accompanied by perfusion, direct injection or local application of the area using other anti-cancer treatments. For example, such treatment may be repeated every 1, 2, 3, 4,5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4,5, 6,7, 8, 9, 10, 11, or 12 months. These treatments may also be at different dosages.

Other types of therapies

Other types of therapies known in the art may be used in combination or together with the methods and compositions provided herein, including but not limited to cryotherapy, hyperthermia therapy, photodynamic therapy, and High Intensity Focused Ultrasound (HIFU) therapy.

Cryotherapy (also known as cryosurgery) uses extreme cold produced by liquid nitrogen (or argon gas) to destroy abnormal tissue. Cryosurgery is used to treat external tumors, such as those on the skin. For external tumors, liquid nitrogen is applied directly to the cancer cells using a swab or spray device. Cryosurgery is also used to treat tumors inside the body (both internal tumors and tumors in the bone). For internal tumors, liquid nitrogen or argon gas is circulated through a hollow device called a cryoprobe in contact with the tumor. The probe may be inserted into the tumor during surgery or through the skin (transdermally). After cryosurgery, the frozen tissue thaws, is either naturally absorbed by the body (for internal tumors), or dissolves and forms a scab (for external tumors).

Hyperthermia (also known as heat therapy or thermotherapy) is a type of cancer treatment in which body tissue is exposed to high temperatures (up to 113 ° F). There are several hyperthermia methods, including local, regional and systemic hyperthermia.

In localized hyperthermia, various techniques of delivering energy to heat a tumor are used, heat is applied to a small area, e.g., a tumor. Different types of energy may be used to apply heat, including microwave, radio frequency, and ultrasound. Depending on the tumor location, there are several methods of localized hyperthermia, including external methods, intraluminal or intraluminal methods, and interstitial techniques.

In regional hyperthermia, a variety of methods are used to heat large areas of tissue, such as body cavities, organs or limbs, including deep tissue methods, local perfusion techniques, and Continuous Hyperthermic Peritoneal Perfusion (CHPP).

Whole body hyperthermia can be used to treat metastatic cancer throughout the body, which can be accomplished with several techniques that raise body temperature to 107-.

Photodynamic therapy (PDT) is a treatment using agents called photosensitizers or photosensitizing agents, as well as certain types of light. When photosensitizers are exposed to light of a particular wavelength, they produce a form of oxygen that kills nearby cells. In the first step of PDT for cancer treatment, a light sensitive agent is injected into the bloodstream. The agent is taken up by cells throughout the body, but remains longer in cancer cells than in normal cells. Approximately 24 to 72 hours after injection, the tumor is exposed to light when most of the agent leaves normal cells but remains in the cancer cells. Photosensitizers in tumors absorb light, producing an active form of oxygen that destroys nearby cancer cells.

The light used for PDT may come from a laser or other source. The laser light can deliver light directly through a fiber optic cable (a thin fiber that transmits light) to an area inside the body. Other light sources include Light Emitting Diodes (LEDs), which can be used for superficial tumors, such as skin cancer. Extracorporeal photopheresis (ECP) is a PDT in which a machine is used to collect blood cells from a patient, treat them with a photosensitizer in vitro, expose them to light, and then return them to the patient.

High intensity focused ultrasound therapy (or HIFU) is one type of cancer treatment. Physicians perform HIFU therapy using machines that emit high frequency sound waves, deliver a strong beam to specific parts of the cancer, and kill cancer cells.

Other agents

It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to enhance the therapeutic efficacy of the treatment. These additional agents include agents that affect the modulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. Increasing intercellular signaling by increasing the number of GAP junctions may increase the anti-hyperproliferative proliferative effect on neighboring hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the present embodiments to enhance the anti-hyperproliferative efficacy of the treatments. Cell adhesion inhibitors are contemplated to improve the efficacy of this example. Examples of cell adhesion inhibitors are inhibitors of Focal Adhesion Kinases (FAKs) and lovastatin. It is also contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of the present embodiments to improve therapeutic efficacy.

5.7 companion diagnostics

The present disclosure is based, at least in part, on the recognition that expression of BTN1a1 and PD-L1 are mutually exclusive in certain cancers. Accordingly, provided herein are methods of using BTN1a1, BTN1a1 ligands (e.g., GAL-1, GAL-9, NRP-2, BTLA), and PD-L1 as biomarkers and concomitant diagnostics for cancer.

BTN1a1 is highly specifically expressed in cancer cells. Also provided herein are methods of detecting expression of BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL9, NRP-2, BTLA) in a sample of a subject using the molecules described herein having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand. Thus, also provided herein is the use of the molecules described herein as a cancer diagnostic agent. In some embodiments, provided herein are methods of detecting BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in a sample from a subject by contacting the sample with a molecule described herein to form a complex between the molecule and BTN1a1 or BTN1a1 ligand, and detecting the complex in the sample. In some embodiments, provided herein are methods of providing or aiding diagnosis of cancer in a subject, comprising contacting a sample from the subject with a molecule described herein to form a complex between the molecule and BTN1a1 or BTN1a1 ligand, detecting the complex, and diagnosing that the subject may have cancer if the complex is detected in the sample. In some embodiments, the methods comprise detecting the presence of glycosylated BTN1a1 in a sample using a molecule described herein having an antigen binding fragment that immunospecifically binds to glycosylated BTN1a 1.

In some embodiments, the method further comprises detecting the presence of PD-L1. Methods for detecting PD-L1 expression in a sample are known in the art and include, for example, antibody-based detection methods (e.g., ELISA, FACS, immunocytochemistry) or nucleic acid-based detection methods (e.g., PCR, microarray, DNA sequencing).

Also provided herein are methods of detecting expression of BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in a sample from a subject using the molecules described herein that competitively block (e.g., in a dose-dependent manner) an antigen binding fragment of a BTN1a1 epitope or a BTN1L1 ligand epitope described herein. In some embodiments, the method further comprises detecting expression of PD-L1.

Also provided herein are methods of detecting expression of BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in a sample from a subject using a molecule described herein having an antigen binding fragment that immunospecifically binds to an epitope of BTN1a1 or BTN1a1 ligand described herein. In some embodiments, the method further comprises detecting expression of PD-L1.

In some embodiments, detecting BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in a sample using a molecule described herein comprises measuring the expression level of BTN1a1 or BTN1a1 ligand in the sample. In some embodiments, detecting BTN1a1 or BTN1a1 ligand further comprises comparing the expression level of BTN1a1 or BTN1a1 ligand in a sample from the subject to a reference level. In some embodiments, the methods comprise measuring the expression level of BTN1a1 or BTN1a1 ligand in the sample using a molecule described herein, comparing the expression level of BTN1a1 or BTN1a1 ligand in the sample to a reference level, and diagnosing that the subject is likely to have cancer or is likely to be responsive to a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand if the expression level of BTN1a1 or BTN1a1 ligand in the sample is higher than the reference level.

In some embodiments, detecting PD-L1 in a sample comprises measuring the expression level of PD-L1 in the sample using a molecule described herein. In some embodiments, detecting PD-L1 further comprises comparing the expression level of PD-L1 in a sample from the subject to a reference level. In some embodiments, the methods comprise measuring the expression level of PD-L1 in the sample using the molecules described herein, comparing the expression level of PD-L1 in the sample to a reference level, and diagnosing that the subject is likely to be responsive to a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) if the expression level of PD-L1 in the sample is below the reference level.

In some embodiments, measuring the level of BTN1a1 or BTN1a1 ligand level comprises measuring the level of glycosylated BTN1a1 using a molecule (e.g., an anti-glycosylated BTN1a1 antibody) having an antigen binding fragment that immunospecifically binds to glycosylated BTN1a 1. In some embodiments, measuring the level of glycosylated BTN1a1 in the sample further comprises comparing the level of glycosylated BTN1a1 in the sample to a reference level, and if the level of glycosylated BTN1a1 in the sample is higher than the reference level, diagnosing that the subject may have cancer or may be responsive to a molecule comprising an antigen binding fragment that immunospecifically binds to glycosylated BTN1a 1.

In some embodiments, measuring the level of BTN1a1 comprises measuring the level of BTN1a1 dimer using a molecule having an antigen binding fragment that immunospecifically binds to BTN1a1 dimer (e.g., an anti-BTN 1a1 dimer antibody). In some embodiments, measuring the level of BTN1a1 dimer in the sample further comprises comparing the level of dimer BTN1a1 in the sample to a reference level, and if the level of BTN1a1 dimer in the sample is above the reference level, diagnosing that the subject may have cancer or may be responsive to a molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1 dimer.

In some embodiments, the reference level may be the expression level of BTN1a1, BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), or PD-L1 in a sample from a healthy individual. In some embodiments, the reference level may be an average or intermediate expression level of BTN1a1, BTN1a1 ligand, or PD-L1 in a sample from a population of healthy individuals. The reference level may also be a cut-off value determined by statistically analyzing the expression level of BTN1a1, BTN1a1 ligand, or PD-L1 from population samples. Statistical methods that can be used to determine such cutoff values are well known in the art. For example, Receiver Operator Characterization (ROC) analysis can be used to determine the reference expression ratio. An overview of ROC analysis can be found in soride, J Clin Pathol,10:1136(2008), incorporated herein by reference in its entirety.

In certain embodiments, the subject may be a healthy subject undergoing a routine health check. In certain embodiments, the healthy subject is at risk for cancer, as determined by the presence of certain risk factors known in the art. Such risk factors include, without limitation, genetic predisposition, personal disease history, family history, lifestyle factors, environmental factors, diagnostic indicators, and the like. In certain embodiments, the subject is asymptomatic. Asymptomatic subjects further include cancer patients who show a mild signal for early diagnosis of cancer, but no other symptoms or discomfort. In certain embodiments, the subject has cancer.

In certain embodiments, the subject is suspected of having cancer. In certain embodiments, the subject has a genetic predisposition to develop cancer or a family history of cancer. In certain embodiments, the subject is exposed to certain lifestyle factors that promote the development of cancer, or the subject exhibits clinical disease manifestations of cancer. In certain embodiments, the subject is a patient undergoing clinical examination to diagnose cancer or assess the risk of developing cancer.

The cancer may be a metastatic cancer. The cancer may be a hematological cancer or a solid tumor. In certain embodiments, the cancer is a hematological cancer selected from leukemia, lymphoma, and myeloma. In certain embodiments, the cancer is a solid tumor selected from breast cancer, lung cancer, thymus cancer, thyroid cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, kidney cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer, both melanoma and non-melanoma skin cancer. The cancer may also be any other type of cancer described herein.

In certain embodiments, the cancer is a cancer resistant or refractory to anti-PD 1 therapy or anti-PD-L1 therapy. In certain embodiments, the cancer is a cancer resistant or refractory to a PD1 therapy. In certain embodiments, the cancer is a cancer resistant or refractory to an anti-PD-L1 therapy. In certain embodiments, the cancer is breast cancer or lung cancer. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is Lewis lung cancer.

In certain embodiments, the subject is untreated. In certain embodiments, the subject is undergoing a cancer treatment (e.g., chemotherapy). In certain embodiments, the subject is in remission. In certain embodiments, the relief is drug-induced. In certain embodiments, the relief is drug-free.

In some embodiments, a method of detecting BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) or PD-L1 comprises obtaining a sample from a subject. The subject may be a human. The subject may be a cancer patient. The sample may be a whole blood sample, a bone marrow sample, a partially purified blood sample, PBMCs, a tissue biopsy, circulating tumor cells, circulating elements such as protein complexes or exosomes. In certain embodiments, the sample is a blood sample. In certain embodiments, the sample is a tissue biopsy.

In some embodiments, the methods provided herein include detecting BTN1a1, BTN1a1 ligand or PD-L1 in a sample using various Immunohistochemical (IHC) methods or other immunoassay methods of the molecules described herein, including anti-BTN 1a1 antibody, anti-BTN 1a1 ligand antibody and anti-PD-L1 antibody.

IHC staining of tissue sections has proven to be a reliable method to assess or detect the presence of proteins in a sample. Immunohistochemical techniques typically utilize antibodies to detect and visualize cellular antigens in situ by chromogenic or fluorescent methods. Thus, antibodies or antisera, preferably polyclonal antisera, most preferably monoclonal antibodies, specific for BTN1a1, BTN1a1 ligands (e.g., GAL-1, GAL-9, NRP-2, BTLA) or PD-L1 can be used. As discussed in more detail below, the antibody can be detected by directly labeling the antibody itself, e.g., using a radioactive label, a fluorescent label, a hapten label such as biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibodies are used in conjunction with labeled secondary antibodies, including antisera, polyclonal antisera, or monoclonal antibodies specific for the primary antibodies. Immunohistochemistry protocols and kits are well known in the art and are commercially available. Automated systems for slide preparation and IHC processing are commercially available.

The BenchMark XT system is an example of such an automated system.

Standard immunological and immunological analysis procedures can be found in Basic and Clinical Immunology (Stits & Terr eds.,7th ed.1991). Furthermore, immunoassays can be performed in any of several configurations, which are described above in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane for extensive review. For an overview of general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37(Asai, ed.1993); basic and Clinical Immunology (Stits & Ten, eds.,7the 1991).

Common assays for detecting BTN1a1, BTN1a1 ligand or PD-L1 include enzyme-linked immunosorbent assay (ELISA), Fluorescent Immunoadsorbent Assay (FIA), chemiluminescent immunoadsorbent assay (CLIA), Radioimmunoassay (RIA), enzyme-multiplied immunoassay (EMI), solid-phase radioimmunoassay (SPROA), Fluorescence Polarization (FP) assay, Fluorescence Resonance Energy Transfer (FRET) assay, time-resolved fluorescence resonance energy transfer (TR-FRET) assay, and Surface Plasmon Resonance (SPR) assay.

In some embodiments, the ELISA is a sandwich ELISA. In some embodiments, the ELISA is a direct ELISA. In some embodiments, the ELISA comprises an initial step of immobilizing the molecules described herein on a solid support (e.g., on the walls of a microtiter plate well or cuvette).

Assays for detecting BTN1A1, BTN1A1 ligand or PD-L1 include non-competitive assays such as sandwich assays and competitive assays. Typically, assays such as ELISA may be used. ELISA assays are known in the art, for example, for assaying various tissues and samples including blood, plasma, serum, or bone marrow.

A number of immunoassay techniques are available utilizing this assay format, see, for example, U.S. patent nos.4,016,043, 4,424,279 and 4,018,653, incorporated herein by reference in their entirety. These include single-site and double-site or "sandwich" (sandwich) assays, which are non-competitive, as well as in traditional competitive binding assays. These assays also include direct binding of labeled antibodies to the target antigen. Sandwich assays are commonly used assays. There are many variations of the sandwich assay technique. For example, in a typical forward assay, an unlabeled anti-BTN 1a1 antibody is immobilized on a solid substrate, and the sample to be tested is contacted with the bound antibody. After a suitable incubation period for a time sufficient to allow formation of an antibody-antigen complex, a second anti-BTN 1a1 antibody specific for the antigen, labeled with a reporter molecule capable of producing a detectable signal, is added and incubated for a time sufficient to allow formation of another complex of antibody-antigen-labeled antibody. Any unreacted material is washed away and the presence of the antigen is confirmed by observing the signal generated by the reporter molecule. The results may be qualitative by simply observing the visible signal, or quantitative by comparison with a control sample containing a standard amount of antigen.

A variant of the forward assay includes a parallel assay (simultaneous assay), in which the sample and labeled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art and will be apparent to include any minor variations. In a typical forward sandwich assay, for example, a primary anti-BTN 1a1 antibody or a primary anti-PD-L1 antibody is covalently or passively bound to a solid surface. The solid surface may be glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid support may be in the form of a test tube, bead, disc of a microplate, or any other surface suitable for performing an immunoassay. The binding process is well known in the art and generally consists of covalent cross-linking binding or physical adsorption, washing the polymer-antibody complex in preparing the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated under suitable conditions (e.g., between room temperature and 40 ℃, e.g., between 25 ℃ and 32 ℃, inclusive) for a time (e.g., 2-40 minutes, or, more conveniently, overnight) sufficient to allow binding of any subunit present in the antibody. After the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the antigen. The second anti-BTN 1a1 antibody or the second anti-PD-L1 antibody is linked to a reporter molecule that is used to indicate binding of the second antibody to a molecular marker.

In some embodiments, flow cytometry (FACS) can be used to detect the level of BTN1a1, BTN1a1 ligand, or PD-L1 in a sample. The intensity of the fluorochrome-labeled antibody, which indicates the level of BTN1a1, BTN1a1 ligand, or PD-L1, was detected and reported by flow cytometry. Non-fluorescent cytoplasmic proteins can also be observed by staining permeabilized cells. The staining agent may be a fluorescent compound capable of binding to certain molecules, or a fluorophore-labeled antibody that binds to a selected molecule.

In the case of enzyme immunoassays, enzymes are typically conjugated to the second antibody via glutaraldehyde or periodate, however, as will be readily appreciated, there are a variety of different conjugation techniques that are readily available to those skilled in the art. enzymes commonly used include horseradish peroxidase, glucose oxidase, β -galactosidase, and alkaline phosphatase, among others.

Thus, provided herein are cancer diagnostic methods comprising detecting the presence or expression level of BTN1a1 or BTN1a1 ligand in a sample from a subject using a molecule having an antigen binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand as described herein. In some embodiments, the method further comprises administering a cancer therapy to the subject diagnosed as having cancer. The cancer treatment can be any cancer therapy described herein or known in the art. In some embodiments, the cancer therapy comprises administering to the subject a therapeutically effective amount of an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand antibody. In some embodiments, the cancer patency comprises administering a therapeutically effective amount of an anti-PD 1 therapy or an anti-PD-L1 therapy.

5.8 evaluation of treatment efficacy

The expression level of BTN1a1 in a subject may be correlated with cancer progression. An increase in the level of BTN1a1 may indicate cancer progression and a decrease in the level of BTN1a1 may indicate cancer regression. Accordingly, the invention also provides methods of using the level of BTN1a1 or the level of BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in a sample of a subject during treatment with a molecule described herein that immunospecifically binds to BTN1a1 or an antigen-binding fragment of BTN1a1 ligand to determine the efficacy of a particular cancer treatment in the subject. In some embodiments, the methods comprise detecting the expression level of BTN1a1 or BTN1a1 ligand. In some embodiments, the methods comprise detecting the level of glycosylated BTN1a 1. In some embodiments, the method comprises detecting the level of BTN1a1 dimer.

In some embodiments, the molecule has an antigen-binding fragment that immunospecifically binds GAL-1, GAL-9, NRP-2, or BTLA. In some embodiments, the molecule has an antigen-binding fragment that immunospecifically binds to GAL-1. In some embodiments, the molecule has an antigen-binding fragment that immunospecifically binds to GAL-9. In some embodiments, the molecule has an antigen-binding fragment that immunospecifically binds to NRP-2. In some embodiments, the molecule has an antigen binding fragment that immunospecifically binds to BTLA.

In some embodiments, the invention also provides methods of assessing the efficacy of a particular cancer treatment in a subject by monitoring the level of BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) levels in a sample of the subject using a molecule described herein having an antigen binding fragment during the course of the treatment. In some embodiments, the molecule can have an antigen binding fragment that competitively blocks (e.g., in a dose-dependent manner) an epitope of BTN1a1 or an epitope of BTN1a1 ligand described herein.

In some embodiments, provided herein are methods of assessing the efficacy of a particular cancer treatment in a patient, comprising a) contacting two or more samples obtained from the patient with a molecule described herein at a first and at least one subsequent point in time throughout the treatment process; b) measuring the level of BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) in two or more samples, and c) comparing the level of BTN1a1 or BTN1a1 ligand in the two or more samples, wherein a decrease in the level of BTN1a1 or BTN1a1 ligand in samples obtained at a subsequent time point relative to the level of BTN1a1 or BTN1a1 ligand in samples obtained at a first time point indicates that the cancer treatment is effective. The molecule may be an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand antibody. In some embodiments, the level of BTN1a1 may be the level of glycosylated BTN1a 1. In some embodiments, the BTN1a1 level can be the level of BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer).

In some embodiments, the method comprises contacting two or more samples obtained from the patient with the molecule described herein at a first and at least one subsequent point in time throughout the course of treatment to form a complex between the molecule and BTN1a1 or BTN1a1 ligand in the samples, and measuring the level of BTN1a1 or BTN1a1 ligand in the two or more samples by measuring the complex in the samples.

In some embodiments, the level of BTN1a1 or BTN1a1 ligand is measured in one assay from two or more samples. In other embodiments, the level of BTN1a1 or BTN1a1 ligand is measured in multiple assays from two or more samples. In some embodiments, the level of BTN1a1 or BTN1a1 ligand is measured on the same day that the sample is obtained from the subject. In some embodiments, the level of BTN1a1 or BTN1a1 ligand is measured without preserving the sample obtained from the subject.

The sample from the cancer patient may be a whole blood sample, a bone marrow sample, a partially purified blood sample, PBMCs, a tissue biopsy, circulating tumor cells, circulating components such as protein complexes or exosomes. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a tissue biopsy. As will be appreciated by those of ordinary skill in the art, any of the methods described herein or otherwise known in the art for determining the level of protein expression in a sample may be used to determine the level of BTN1a1 or BTN1a1 ligand in a sample from a cancer patient. In some embodiments, the method comprises an immunoassay. The immunoassay may be an immunohistochemical method comprising using the molecules described herein to detect and visualize BTN1a1 or BTN1a1 ligand. The immunoassay may comprise a FIA, CLIA, RIA, EMI, SPROA, FP assay, FRET assay, TR-FRET assay or SPR assay.

The cancer treatment or cancer therapy may be any therapy described herein or known in the art, including but not limited to: surgical therapy, chemotherapy, biological targeted therapy, small molecule targeted therapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, and cytokine therapy. In certain embodiments, the cancer treatment comprises an FDA-approved cancer treatment, including experimental cancer treatments in clinical development. In certain embodiments, the cancer treatment comprises treatment with two or more drugs, or a combination of two or more types of therapy.

In some embodiments, the cancer treatment comprises administering to a cancer patient an anti-BTN 1a1 antibody or a BTN1a1 ligand antibody.

In some embodiments, one or more samples are obtained at the beginning of a cancer treatment session and one or more samples are obtained at a subsequent time point throughout the treatment session. In some embodiments, the subsequent time points are 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, or 30 or more time points.

In certain embodiments, if the treatment is determined to be ineffective, the method further comprises adjusting the treatment. Adjusting the treatment can include, for example, adjusting a dosage of the medication, providing a frequency of the medication, treating with a different medication or combination of medications, or ending the treatment.

In certain embodiments, if the treatment is determined to be effective, the method further comprises repeating the treatment.

In some embodiments, the level of BTN1a1 or BTN1a1 ligand in the sample obtained at the first time point is reduced by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or more than 99% from the level at the subsequent time point.

5.9 patient selection

Provided herein are methods of predicting the responsiveness of a cancer patient to a cancer therapy by detecting the presence or expression level of BTN1a1 or BTN1a1 ligand in a patient sample using a molecule having an antigen binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-2, NRP-2, BTLA). In some embodiments, the method comprises detecting BTN1a1 or BTN1a1 ligand in a sample from a cancer patient by contacting the sample with a molecule described herein to form a complex between the molecule and BTN1a1 or BTN1a1 ligand, and predicting that the subject is likely to respond to the cancer treatment if the complex is detected. In some embodiments, the methods include detecting the presence of glycosylated BTN1a1 in a sample using a molecule having an antigen binding fragment that immunospecifically binds to glycosylated BTN1a 1. In some embodiments, the methods include detecting the presence of BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer) in a sample using a molecule having an antigen binding fragment that immunospecifically binds to BTN1a1 dimer (e.g., glycosylated BTN1a1 dimer). In some embodiments, the method further comprises determining the presence or expression level of PD-L1. In some embodiments, the methods comprise detecting the presence of a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA) in a sample using a molecule having an antigen binding fragment that specifically binds GAL-1, GAL-9, NRP-2, or BTLA. In some embodiments, the methods comprise detecting the presence of a BTN1a1 ligand, e.g., GAL-1, in a sample using a molecule having an antigen binding fragment that immunospecifically binds to GAL-1. In some embodiments, the methods comprise detecting the presence of a BTN1a1 ligand, e.g., GAL-9, in a sample using a molecule having an antigen binding fragment that immunospecifically binds to GAL-9. In some embodiments, the methods comprise detecting the presence of a BTN1A1 ligand, e.g., NRP-2, in a sample using a molecule having an antigen binding fragment that immunospecifically binds to NRP-2. In some embodiments, the methods comprise detecting the presence of a BTN1a1 ligand, e.g., BTLA, in a sample using a molecule having an antigen binding fragment that immunospecifically binds BTLA.

In other embodiments, detecting BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), e.g., alone or in combination with PD-L1, in a sample comprises measuring the expression level of BTN1a1 or BTN1a1 ligand in the sample using a molecule described herein, e.g., alone or in combination with PD-L1. In some embodiments, detecting BTN1a1 or BTN1a1 ligand, e.g., alone or in combination with PD-L1, further comprises comparing the level of BTN1a1 or BTN1a1 ligand expression in a sample from the subject to a reference level, e.g., alone or in combination with PD-L1. In some embodiments, the methods comprise measuring the expression level of BTN1a1 or BTN1a1 ligand in a sample using an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand antibody, e.g., alone or in combination with PD-L1, e.g., comparing the expression level of BTN1a1 or BTN1a1 ligand, alone or in combination with PD-L1, in a reference sample, and predicting that the subject is likely to be responsive to cancer treatment if the expression level of BTN1a1 or BTN1a1 ligand in the sample is higher than the reference level of BTN1a1 or BTN1a1 ligand, and optionally if the expression level of PD-L1 is lower than the reference level of PD-L1.

In some embodiments, measuring the level of BTN1a1 or BTN1a1 ligand level, e.g., alone or in combination with PD-L1, includes measuring the level of GAL-1, GAL-9, NRP-2, or BTLA using an anti-GAL-1 antibody, an anti-GAL-9 antibody, an anti-NRP-2 antibody, or an anti-BTLA antibody, e.g., alone or in combination with PD-L1. In some embodiments, measuring the level of BTN1a1 or BTN1a1 ligand, e.g., alone or in combination with PD-L1, comprises measuring the level of GAL-1 using an anti-GAL-1 antibody, e.g., alone or in combination with PD-L1. In some embodiments, measuring the level of BTN1a1 or BTN1a1 ligand, e.g., alone or in combination with PD-L1, comprises measuring the level of GAL-9 using an anti-GAL-9 antibody, e.g., alone or in combination with PD-L1. In some embodiments, measuring the level of BTN1a1 or BTN1a1 ligand, e.g., alone or in combination with PD-L1, comprises measuring the level of NRP-2 using an anti-NRP-2 antibody, e.g., alone or in combination with PD-L1. In some embodiments, measuring the level of BTN1a1 or BTN1a1 ligand level, e.g., alone or in combination with PD-L1, comprises measuring the level of BTLA using an anti-BTLA antibody, e.g., alone or in combination with PD-L1.

The sample from the cancer patient may be a whole blood sample, a bone marrow sample, a partially purified blood sample, PBMCs, a tissue biopsy, circulating tumor cells, circulating components such as protein complexes or exosomes. In some embodiments, the sample is a blood sample. Methods of detecting the presence of BTN1a1, BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA) or PD-L1 or measuring the expression level of BTN1a1, BTN1a1 ligand or PD-L1, or methods known in the art, are described herein.

In some embodiments, the cancer treatment comprises administering an anti-BTN 1a1 antibody to a cancer patient. In some embodiments, the cancer treatment further comprises administration of an anti-PD-1 therapy or an anti-PD-L1 therapy.

5.10 kits

Provided herein are kits containing a molecule described herein and one or more auxiliary agents. In certain embodiments, provided herein are kits for the preparation and/or administration of the therapies provided herein. The kit may have one or more sealed vials containing any of the pharmaceutical compositions described herein. The kit may comprise: for example, molecules having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA), and reagents for making, formulating, and/or administering the molecules or performing one or more steps of the methods disclosed herein.

In some embodiments, the antigen binding fragment immunospecifically binds to a BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, or BTLA). In some embodiments, the antigen binding fragment immunospecifically binds to GAL-1. In some embodiments, the antigen binding fragment immunospecifically binds to GAL-9. In some embodiments, the antigen binding fragment immunospecifically binds to NRP-2. In some embodiments, the antigen binding fragment immunospecifically binds BTLA.

In some embodiments, the molecule is an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand antibody. In some embodiments, the anti-BTN 1a1 antibody or anti-BTN 1a1 ligand antibody is a humanized or human antibody.

In some embodiments, the kit further comprises a second anti-cancer agent. The second anticancer agent may be a chemotherapeutic agent, an immunotherapeutic agent, a hormonal therapeutic agent or a cytokine.

In some embodiments, the second anti-cancer agent is an anti-PD-1 therapy or an anti-PD-L1 therapy. In some embodiments, the second anti-cancer agent is an anti-PD-1 therapy. In some embodiments, the second anti-cancer agent is an anti-PD-L1 therapy. In some embodiments, the second anti-cancer agent is an anti-PD 1 therapy and an anti-PD-L1 therapy. In some embodiments, the anti-PD-1 therapy or anti-PD-L1 therapy comprises an anti-PD-1 or anti-PD-L1 antibody or antibody fragment, or a soluble PD-1 or PD-L1 ligand, or an Fc-fusion protein thereof. In some embodiments, the anti-PD-1 therapy comprises nivolumab (Opdivo), pembrolizumab (Keytruda), pidilizumab, AMP-514, or AMP-224. In some embodiments, the anti-PD-1 therapy comprises an anti-PD-1 antibody provided in international application PCT/US 20126/64394. In some embodiments, the anti-PD-L1 therapy comprises yw243.55.s70, MPDL3280A, MEDI-4736, MSB-0010718C or MDX-1105. In some embodiments, the anti-PD-L1 treatment comprises an antibody provided in international application No. PCT/US20106/024691, disclosed as No. wo20106/160792, and international application PCT/US 2017/024027.

Also provided herein are kits useful as companion diagnostics for cancer. In some embodiments, the kit may be used to provide or assist in cancer diagnosis. In some embodiments, the kit can be used to evaluate the efficacy of a cancer treatment. In some embodiments, the kit can be used to predict the responsiveness of a patient to a cancer treatment. In some embodiments, the kit can be used to select patients for a particular cancer treatment. The kit may include, for example, reagents for detecting BTN1a1 or BTN1a1 ligand in a sample. In some embodiments, the kit includes reagents for detecting PD-L1 (e.g., an anti-PD-L1 antibody).

The agent can be a molecule having an antigen-binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BTLA). In some embodiments, the antigen binding fragment binds GAL-1. In some embodiments, the antigen binding fragment binds GAL-9. In some embodiments, the antigen binding fragment binds to NRP-2. In some embodiments, the antigen binding fragment binds to BTLA.

The cancer therapy may be any therapy described herein or known in the art, including but not limited to: surgical therapy, chemotherapy, biological targeted therapy, small molecule targeted therapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, and cytokine therapy. In certain embodiments, the cancer therapy comprises administering to a cancer patient a molecule described herein having an antigen binding fragment that immunospecifically binds to BTN1a1 or BTN1a1 ligand, e.g., an anti-BTN 1a1 antibody or an anti-BTN 1a1 ligand antibody, including anti-GAL-1 antibody, anti-GAL-9 antibody, anti-NRP-2 antibody, and anti-BTLA antibody.

In certain embodiments, the auxiliary reagent of the diagnostic kit may be a secondary antibody, a detection reagent, a fixation buffer, a blocking buffer, a wash buffer, a detection buffer, or any combination thereof.

The secondary antibody may include, for example, an anti-human IgA antibody, an anti-human IgD antibody, an anti-human IgE antibody, an anti-human IgG antibody, or an anti-human IgM antibody. In certain embodiments, the secondary antibody is an anti-bovine antibody. The second detection antibody may be a monoclonal antibody or a polyclonal antibody. The secondary antibody may be from any mammalian organism, including mouse, rat, hamster, goat, camel, chicken, rabbit, and others. The secondary antibody can be conjugated to an enzyme (e.g., horseradish peroxidase (HRP), Alkaline Phosphatase (AP), luciferase, etc.) or a dye (e.g., a colorimetric dye, a fluorescent dye, a Fluorescence Resonance Energy Transfer (FRET) -dye, a time-resolved (TR) -FRET dye, etc.). In certain embodiments, the secondary antibody is a polyclonal rabbit anti-human IgG antibody, which is HRP conjugated.

In certain embodiments, the detection reagent comprises a fluorescent detection reagent or a luminescent detection reagent. In certain other embodiments, the luminescence detection reagent comprises luminol or luciferin.

A large selection of wash buffers is known in the art, for example Tris (hydroxymethyl) aminomethane (Tris) based buffers (e.g., Tris-buffered saline, TBS) or phosphate buffers (e.g., phosphate buffered saline, PBS). The wash buffer may include a detergent, for example, an ionic or non-ionic detergent. In certain embodiments, the wash buffer is a buffer comprising

(e.g., about 0.05%

) E.g., about pH 7.4.

Any dilution buffer known in the art may be included in the kits of the present disclosure. The dilution buffer can include a carrier protein (e.g., bovine serum albumin, BSA) and a detergent (e.g.,

). In certain embodiments, the dilution buffer is a buffer comprising BSA (e.g., about 1% BSA) and

(e.g., about 0.05%

) E.g., about pH 7.4.

In certain embodiments, the detection reagent is a colorimetric detection reagent, a fluorescent detection reagent, or a chemiluminescent detection reagent. In certain embodiments, the colorimetric detection reagent comprises PNPP (p-nitrophenyl phosphate), ABTS (2,2' -azidobis (3-ethylbenzothiazoline-6-sulfonic acid)), or OPD (o-phenylenediamine). In certain embodiments, the fluorescent detection reagent comprises QuantaBlu or QuantaRedTM (Thermo Scientific, Waltham, Mass.). In some embodiments, the luminescence detection reagent comprises luminol or luciferin. In certain embodiments, the detection reagent comprises a trigger (e.g., H)₂O₂) And a tracer (e.g., isoluminol-conjugate).

Any detection buffer known in the art may be included in the kits of the present disclosure. In certain embodiments, the detection buffer is a citrate-phosphate buffer (e.g., about ph 4.2).

Any termination solution known in the art may be included in the kits of the present disclosure. The stop solutions of the present disclosure stop or delay further development of the detection reagents and corresponding analytical signals. The termination solution can include, for example, a low pH buffer (e.g., glycine buffer, pH 2.0), a chaotropic agent (e.g., guanidinium chloride, Sodium Dodecyl Sulfate (SDS)), or a reducing agent (e.g., dithiothreitol, mercaptoethanol), among others.

In certain embodiments, the kits provided herein comprise cleaning reagents for an automated analysis system. The automated analysis system may comprise any vendor's system. In certain embodiments, the automated analysis system includes, for example, BIO-FLASHTM, BEST 2000TM, DS2TM, ELx50 WASHER, ELx800READER, and Autoblot S20 TM. The cleaning agent may comprise any cleaning agent known in the art. In certain embodiments, the cleaning reagent is a manufacturer recommended cleaning reagent for the automated analysis system.

In certain embodiments, the kit may further comprise a suitable container means which is a container that is not reactive with the components of the kit, e.g., an Eppendorf tube, an assay plate, a syringe, a bottle, or a test tube. The container may be made of a sterilizable material such as plastic or glass.

In certain embodiments, the kit further comprises a solid support. The solid support may comprise any support known in the art on which a protein of the present disclosure may be immobilized. In certain embodiments, the solid substrate is a microtiter well plate, a slide (e.g., a glass slide), a chip (e.g., a protein chip, a biosensor chip, e.g., a Biacore chip), a microfluidic cartridge, a cuvette, a bead (e.g., a magnetic bead), or a resin.

In some other embodiments, the kits provided herein include instructions for using the subunits of the kit to detect BTN1a1 or BTN1a1 ligand (e.g., GAL-1, GAL-9, NRP-2, BLTA) in a sample from the subject.

The kits provided herein may be adapted for a particular analytical technique. In certain embodiments, the kit is an ELISA kit, a dot blot kit, a Chemiluminescent Immunoassay (CIA) kit, or a multiplex kit. In certain embodiments, the ELISA kit can include a wash buffer, a sample diluent, a secondary antibody-enzyme conjugate, a detection reagent, and a stop solution. In certain embodiments, the dot blot kit can include a wash buffer, a sample diluent, a secondary antibody-enzyme conjugate, a detection reagent, and a stop solution. In certain embodiments, the CIA kit comprises a wash buffer, a sample diluent, a tracer (e.g., isoluminol-conjugate), and a trigger (e.g., H)₂O₂). In certain embodiments, the multiplex kit comprises a wash buffer, a sample diluent, and a secondary antibody-enzyme conjugate.

In certain embodiments, the kits of the invention have a package comprising a label indicating that the kit is for diagnosis, prognosis, or monitoring of cancer. In certain embodiments, the kit is used as a companion diagnostic for cancer therapy. In certain other embodiments, the package has a label indicating that the kit is for use with a cancer drug. In certain embodiments, the kit is used to select patients for a particular cancer treatment.

In certain embodiments, the package of the kit comprises an FDA approved label. The FDA approved label may include a notice and instructions for FDA approved use. In certain embodiments, the kit is labeled for Research Use Only (RUO) or Research Use Only (IUO). In certain embodiments, the kit is labeled for In Vitro diagnosis (In Vitro Diagnostic Use, IVD). In certain embodiments, the kit is labeled according to title 21 of federal regulations, section 809, part B (21CFR 89, part B).

6. Examples of the embodiments

It is to be understood that modifications are also contemplated which do not materially alter the nature and spirit of the various embodiments described herein. Accordingly, the following examples are intended to be illustrative, but not limiting in any way.

6.1 example 1 galectin-1, galectin-9, neuropilin 2 as BTN1A1 ligand Identification of

To identify BTN1A1 ligands, Retrogenix was used^TMCell microarray technology (Whaley Bridge, United Kingdom) screens arrays of plasma membrane proteins.

Briefly, expression vectors encoding plasma membrane proteins were arrayed on slides and reverse transfected into HEK293 cells to generate cell microarrays expressing candidate BTN1a1 ligands. Binding of BTN1A1-Fc to the candidate BTN1A1 ligand was detected by fluorescence imaging. Using the same cell microarray technique and secondary analysis, including co-immunoprecipitation and surface plasmon resonance analysis (Biacore)^TM) The identified BTN1a1 ligand was confirmed.

Prescreening and reconfirming

4550 expression vectors, each encoding a full-length human plasma membrane protein, were arranged in duplicate on 13 microarray slides ("slide sets"). 3500 multiple arrays of expression vectors encode unique genes. One expression vector control (pIRES-hEGFR-IRES-ZsGreen1) was spotted on each slide at four-fold (quadruplicates) to confirm that the minimum threshold for transfection efficiency was met or exceeded on each slide (average ZsGreen signal from pIRES-hEGFR-ZsGreen vector was 1.5 above background). Human HEK293 cells were used for reverse transfection and protein expression. BTN1A1-ECD-Fc was added as a test ligand to each slide at a final concentration of 20. mu.g/ml after cell fixation. HEK293 cells expressing BTN1a1 ligand bound to BTN1a1-Fc or negative control cells were detected using anti-IgG antibodies and fluorescence imaging. Two replicate slides were screened for each of the 13 microarray slides of the primary screening slide set. The fluorescence images were analyzed and quantified (for transfection efficiency) using ImageQuant software (GE). Primary "hits" were defined as duplicate spots on the microarray slide that showed increased signal relative to background levels. Primary hits were identified by visual inspection of the images gridded by ImageQuant software. Based on the fluorescence intensity of the repeated spots, the primary hits were classified as "strong", "moderate", "weak" or "very weak".

Primary hits were again confirmed in cell microarray analysis. The vectors encoding the primary hits are arranged on multiple slide sets. Each slide in the confirmation assay also included a CD86 positive control and an EGFR negative control. The candidate BTN1a 1-ligand was expressed in HEK293 cells and the cells were fixed. Different slides (n-2 slides per treatment) were then treated with 20 μ g/ml btn1a1-Fc, 20 μ g/ml ctla4-Fc or no ligand (antibody only detected).

BTN1A1-Fc typically only showed low background binding to immobilized untransfected HEK293 when tested at 2. mu.g/ml, 5. mu.g/ml or 20. mu.g/ml. A total of 40 slides were screened in the primary screen (26 slides) and secondary confirmation assay (14 slides). The transfection efficiency exceeds the target transfection efficiency threshold. In the initial screen, 11 repeated hits (expression vector clones) were identified. The 11 primary hits covered a range of spot intensities (signal against background) from very weak to strong. The identified vectors were sequenced to confirm the identity of the candidate BTN1a1 ligand. Vectors encoding 11 hits, either CD86/ZsGreen1 (positive control) or EGFR/ZsGreen1 (negative control), were tested in a double confirmation assay.

Fig. 1 shows an image illustrating the results of an exemplary reconfirmation assay. A strong binding signal was observed for CTLA4-hFc (soluble ligand) interaction with the positive control of CD86 (cell membrane protein), confirming the assay conditions. In the reconfirmed assay, three primary hits, galectin-1 (Gal-1), galectin-9 (Gal-9) and neuropilin-2 (NRP-2), were shown to bind specifically to BTN1Al-Fc, but not to the negative control. Thus, GAL-1, GAL-9 and Nrp2 were shown to be BTN1A ligands.

Co-immunoprecipitation

Gal-1 and Gal-9 were further confirmed to be BTN1A1 ligands by co-immunoprecipitation (co-IP). BTN1A1-Flag was expressed in HEK293T cells, alone or in combination with Myc-and Flag-ditag-1 or Gal-9 (FIG. 2A). Immunoprecipitation was then performed with anti-Myc or anti-BTN 1a1 antibodies (pull-down) (fig. 2B). The immunoprecipitates were analyzed by SDS-PAGE to detect the interaction between BTN1A1 and Gal-1 or Gal-9 (FIG. 2C). Pull-down of Myc-tagged Gal-1 and Gal-9 with anti-Myc antibody resulted in co-IP of Flag-tagged BTN1A1 (FIG. 2C). To further confirm the specificity of the interaction between BTN1A1-Gal-1 and BTN1A1-Gal9, BTN1A1 was pulled down using BTN1A 1-specific antibody STC 810. Pulling down BTN1A1 with STC810 resulted in co-IP for Gal-1 and Gal-9 (FIG. 2C). These results further confirmed that Gal-1 and Gal-9 are authentic ligands for BTN1A 1.

Surface plasmon resonance

In surface plasmon resonance (Biacore)^TM) The BTN1A1-Gal-1 interaction was further analyzed in a binding assay. The BTN1a1-Fc fusion protein was captured on a protein a coated CM5 chip and GAL-1 ligand was injected at five or more different concentrations onto a CM5 chip. FIGS. 3A-D show exemplary BIAcore sensorgrams illustrating binding of Gal-1-to either glycosylated BTN1A1 wild-type (BTN1A1, FIG. 3A), non-glycosylated BTN1A1 mutant (BTN1A1-2NQ) or other members of the BTN1A1 family (BTN2A 1-FIG. 3C; BTN3A 2-FIG. 3D).

No detectable binding of Gal-1 to the nonglycosylated BTN1A1-2NQ mutein was found (FIG. 3B), demonstrating that Gal-1 binds BTN1A1 in a carbohydrate-specific manner. In addition, Gal-1 was not found to detectably bind other members of the BTN1A1 family, such as BTN2A1 (FIG. 3C) or BTN3A2 (FIG. 3D), demonstrating that Gal-1 is a selective ligand for BTN1A1 in the BTN1A1 family.

6.2 example 2 identification of BLTA as a ligand for BTN1A1

β -galactosidase complementation assay

The β -Gal activity resulting from the forced interaction of non-functional weakly complementary β -Gal peptides (Δ α and Δ ω) can be used as a measure of the degree of interaction of the non- β -Gal portion of the chimera with β -Gal complementation, cis-acting protein-protein interactions occurring at the plasma membrane can be detected.

FIG. 4A shows a diagram illustrating the design of β -Gal complementation assay β -galactosidase complementation assay is usually designed using two inactive fragments of β -galactosidase (β -Gal), Enzyme Donor (ED) and enzyme receptor (EA) that bind to produce active β -Gal enzyme, the N-terminal 45 amino acid fragment pro-linker (ProLinker, PK) as ED is fused to the candidate BTN1A1 ligand protein, and the interaction between BTN1A1 and EA fusion.the complementation between the two proteins can promote complementation between PK and EA, resulting in the formation of active β -Gal enzyme that cleaves the substrate to produce a chemiluminescent signal, BTN1A1-EA cell line is engineered to stably express large β -Gal enzyme reporter fragment EA (enzyme receptor). the PK fused PK N1A 1-ligand candidate is transfected into EA cell line.3634- β enzyme activity is measured as a direct interaction between N1A 961-Gal 1A1-Gal ligand and BTN 2-Gal ligand 1. BTN1A1-Gal 3-Gal is shown to interact with BTN 11-Gal ligand 39596 enzyme.

Co-immunoprecipitation

To confirm that BTN1A1 ligand identified in the β -Gal complementation screen, BTN1A1 was Co-transfected with BTLA and Nrp-2 and Co-immunoprecipitated FIG. 5 shows a Western blot image with representative results of the BTN1A1-BTLACo-IP experiment Co-IP with anti-FLAG or anti-Myc antibody, as shown in FIG. 5 precipitation of HEK293T cells expressing BTN1A1-FLAG and BTLA-Myc or Nrp-2-Myc was analyzed by SDS-PAGE (Nrp-2 data not shown in FIG. 5). Co-IP with anti-FLAG antibody resulted in Co-precipitation of BTLA-Myc or Nrp-2-Myc. to confirm the specificity of the observed interaction of BTN1A 567-Myc-N1A 1 ligand, Co-precipitation of BTN1A1 with BTN-Myc or Nrp-Myc was found by pulling down from the cell lysate using anti-FLAC antibody.

Surface plasmon resonance

In Biacore^TMThe interaction between BTN1a1 and BTLA was further analyzed in the assay. The BTN1A1-Fc fusion protein was captured on a protein A-coated CM5 chip and BTLA or Gal-1 was injected at 3.2. mu.M onto the chip. FIG. 6A shows a sensorgram illustrating binding of Gal-1 and BTLA to BTN1A 1-Fc. Gal-1 binding is indicated by a positive reaction signal in the sensorgram. BTLA and Biacore^TMImmobilized BTN1A1-Fc binding on the chip resulted in a decrease in the response signal (negative signal) in the sensorgram. FIG. 6B shows Biacore with immobilized BTN1A1-Fc at 0.4. mu.M, 0.8. mu.M, 6. mu.M, 3.2. mu.M, or 6.4. mu.M when BTLA is injected^TMA sensorgram of dose-dependent reduction in response signals observed while on-chip. Without being bound by a particular theory, it is believed that the observed decrease in SPR signal caused by BTLA binding to BTN1a1 indicates a conformational change, such as BTN1a1 conformational change upon BTN1a1-BTLA complex formation.

Biological layer interferometry

FIG. 7 shows a sensorgram illustrating the results of a biolayer interferometry (BLI) experiment at

BTLA was analyzed on a system (Fortebio, Menlo Park, Calif.) for binding to BTN1A 1-Fc. Coating with BTN1A1-Fc

anti-hIgG capture (AHC) end of the system. Biosensor to be coatedThe ends were immersed in increasing concentrations of BTLA (0.8. mu.M, 1.6. mu.M or 3.2. mu.M) solution and binding of BTLA to BTN1A1 was monitored. Table XX shows the results of the binding kinetics and dissociation constants of the BTN1A1-BTLA interaction determined separately for each BTN1A1 concentration. Total K was calculated by steady state analysis using ForteBio data analysis 9.0 software_DThe value is obtained. From the calculation, K is determined for BTLA binding to BTN1A1_D17 μ M. + -. 0.81 μ M.

Sample loading ID

Sample ID

Concentration (μ M)

Response to

K_D(M)

k_on(1/Ms)

k_dis(1/s)

RMax

BTN1A1-Fc

BTLA-His

6.4

0.0840

8.43E-07

9.23E+02

7.78E-04

0.0849

BTN1A1-Fc

BTLA-His

3.2

0.0436

5.79E-07

1.26E+03

7.28E-04

0.0476

BTN1A1-Fc

BTLA-His

1.6

0.0227

7.24E-07

1.65E+03

1.19E-03

0.0336

BTN1A1-Fc

BTLA-His

0.8

0.0067

9.53E-06

7.98E+01

7.61E-04

0.2222

***

Throughout this application, various publications have been referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this disclosure pertains. While examples of certain specific embodiments are provided herein, it will be apparent to those skilled in the art that various changes and modifications can be made. Such modifications are also intended to fall within the scope of the appended claims.

Sequence listing

<110> Stateku corporation

<120> methods of treating cancer using antibodies and molecules that bind BTN1A1 or BTN1A 1-ligand

<130>13532-020-228

<140>TBA

<141>

<150>62/516,071

<151>2017-06-06

<160>14

<170>PatentIn version 3.5

<210>1

<211>526

<212>PRT

<213> Intelligent people

<400>1

Met Ala Val Phe Pro Ser Ser Gly Leu Pro Arg Cys Leu Leu Thr Leu

1 5 10 15

Ile Leu Leu Gln Leu Pro Lys Leu Asp Ser Ala Pro Phe Asp Val Ile

20 25 30

Gly Pro Pro Glu Pro Ile Leu Ala Val Val Gly Glu Asp Ala Lys Leu

35 40 45

Pro Cys Arg Leu Ser Pro Asn Ala Ser Ala Glu His Leu Glu Leu Arg

50 55 60

Trp Phe Arg Lys Lys Val Ser Pro Ala Val Leu Val His Arg Asp Gly

65 70 75 80

Arg Glu Gln Glu Ala Glu Gln Met Pro Glu Tyr Arg Gly Arg Ala Thr

85 90 95

Leu Val Gln Asp Gly Ile Ala Lys Gly Arg Val Ala Leu Arg Ile Arg

100 105 110

Gly Val Arg Val Ser Asp Asp Gly Glu Tyr Thr Cys Phe Phe Arg Glu

115 120 125

Asp Gly Ser Tyr Glu Glu Ala Leu Val His Leu Lys Val Ala Ala Leu

130 135 140

Gly Ser Asp Pro His Ile Ser Met Gln Val Gln Glu Asn Gly Glu Ile

145 150 155 160

Cys Leu Glu Cys Thr Ser Val Gly Trp Tyr Pro Glu Pro Gln Val Gln

165 170 175

Trp Arg Thr Ser Lys Gly Glu Lys Phe Pro Ser Thr Ser Glu Ser Arg

180 185 190

Asn Pro Asp Glu Glu Gly Leu Phe Thr Val Ala Ala Ser Val Ile Ile

195 200 205

Arg Asp Thr Ser Ala Lys Asn Val Ser Cys Tyr Ile Gln Asn Leu Leu

210 215 220

Leu Gly Gln Glu Lys Lys Val Glu Ile Ser Ile Pro Ala Ser Ser Leu

225 230 235 240

Pro Arg Leu Thr Pro Trp Ile Val Ala Val Ala Val Ile Leu Met Val

245 250 255

Leu Gly Leu Leu Thr Ile Gly Ser Ile Phe Phe Thr Trp Arg Leu Tyr

260 265 270

Asn Glu Arg Pro Arg Glu Arg Arg Asn Glu Phe Ser Ser Lys Glu Arg

275 280 285

Leu Leu Glu Glu Leu Lys Trp Lys Lys Ala Thr Leu His Ala Val Asp

290 295 300

Val Thr Leu Asp Pro Asp Thr Ala His Pro His Leu Phe Leu Tyr Glu

305 310 315 320

Asp Ser Lys Ser Val Arg Leu Glu Asp Ser Arg Gln Lys Leu Pro Glu

325 330 335

Lys Thr Glu Arg Phe Asp Ser Trp Pro Cys Val Leu Gly Arg Glu Thr

340 345 350

Phe Thr Ser Gly Arg His Tyr Trp Glu Val Glu Val Gly Asp Arg Thr

355 360 365

Asp Trp Ala Ile Gly Val Cys Arg Glu Asn Val Met Lys Lys Gly Phe

370 375 380

Asp Pro Met Thr Pro Glu Asn Gly Phe Trp Ala Val Glu Leu Tyr Gly

385 390 395 400

Asn Gly Tyr Trp Ala Leu Thr Pro Leu Arg Thr Pro Leu Pro Leu Ala

405 410 415

Gly Pro Pro Arg Arg Val Gly Ile Phe Leu Asp Tyr Glu Ser Gly Asp

420 425 430

Ile Ser Phe Tyr Asn Met Asn Asp Gly Ser Asp Ile Tyr Thr Phe Ser

435 440 445

Asn Val Thr Phe Ser Gly Pro Leu Arg Pro Phe Phe Cys Leu Trp Ser

450 455 460

Ser Gly Lys Lys Pro Leu Thr Ile Cys Pro Ile Ala Asp Gly Pro Glu

465 470 475 480

Arg Val Thr Val Ile Ala Asn Ala Gln Asp Leu Ser Lys Glu Ile Pro

485 490 495

Leu Ser Pro Met Gly Glu Asp Ser Ala Pro Arg Asp Ala Asp Thr Leu

500 505 510

His Ser Lys Leu Ile Pro Thr Gln Pro Ser Gln Gly Ala Pro

515 520 525

<210>2

<211>1581

<212>DNA

<213> Intelligent people

<400>2

atggcagttt tcccaagctc cggtctcccc agatgtctgc tcaccctcat tctcctccag 60

ctgcccaaac tggattcagc tccctttgac gtgattggac ccccggagcc catcctggcc 120

gttgtgggtg aggacgccaa gctgccctgt cgcctgtctc cgaacgcgag cgccgagcac 180

ttggagctac gctggttccg aaagaaggtt tcgccggccg tgctggtgca tagggacggg 240

cgcgagcagg aagccgagca gatgcccgag taccgcgggc gggcgacgct ggtccaggac 300

ggcatcgcca aggggcgcgt ggccttgagg atccgtggcg tcagagtctc tgacgacggg 360

gagtacacgt gctttttcag ggaggatgga agctacgaag aagccctggt gcatctgaag 420

gtggctgctc tgggctctga ccctcacatc agtatgcaag ttcaagagaa tggagaaatc 480

tgtctggagt gcacctcagt gggatggtac ccagagcccc aggtgcagtg gagaacttcc 540

aagggagaga agtttccatc tacatcagag tccaggaatc ctgatgaaga aggtttgttc 600

actgtggctg cttcagtgat catcagagac acttctgcga aaaatgtgtc ctgctacatc 660

cagaatctcc ttcttggcca ggagaagaaa gtagaaatat ccataccagc ttcctccctc 720

ccaaggctga ctccctggat agtggctgtg gctgtcatcc tgatggttct aggacttctc 780

accattgggt ccatattttt cacttggaga ctatacaacg aaagacccag agagaggagg 840

aatgaattca gctctaaaga gagactcctg gaagaactca aatggaaaaa ggctaccttg 900

catgcagttg atgtgactct ggacccagac acagctcatc cccacctctt tctttatgag 960

gattcaaaat ctgttcgact ggaagattca cgtcagaaac tgcctgagaa aacagagaga 1020

tttgactcct ggccctgtgt gttgggccgt gagaccttca cctcaggaag gcattactgg 1080

gaggtggagg tgggagacag gactgactgg gcaatcggcg tgtgtaggga gaatgtgatg 1140

aagaaaggat ttgaccccat gactcctgag aatgggttct gggctgtaga gttgtatgga 1200

aatgggtact gggccctcac tcctctccgg acccctctcc cattggcagg gcccccacgc 1260

cgggttggga ttttcctaga ctatgaatca ggagacatct ccttctacaa catgaatgat 1320

ggatctgata tctatacttt ctccaatgtc actttctctg gccccctccg gcccttcttt 1380

tgcctatggt ctagcggtaa aaagcccctg accatctgcc caattgctga tgggcctgag 1440

agggtcacag tcattgctaa tgcccaggac ctttctaagg agatcccatt gtcccccatg 1500

ggggaggact ctgcccctag ggatgcagac actctccatt ctaagctaat ccctacccaa 1560

cccagccaag gggcacctta a 1581

<210>3

<211>443

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400>3

Ala Pro Phe Asp Val Ile Gly Pro Pro Glu Pro Ile Leu Ala Val Val

1 5 10 15

Gly Glu Asp Ala Glu Leu Pro Cys Arg Leu Ser Pro Asn Ala Ser Ala

20 25 30

Glu His Leu Glu Leu Arg Trp Phe Arg Lys Lys Val Ser Pro Ala Val

35 40 45

Leu Val His Arg Asp Gly Arg Glu Gln Glu Ala Glu Gln Met Pro Glu

50 55 60

Tyr Arg Gly Arg Ala Thr Leu Val Gln Asp Gly Ile Ala Lys Gly Arg

65 70 75 80

Val Ala Leu Arg Ile Arg Gly Val Arg Val Ser Asp Asp Gly Glu Tyr

85 90 95

Thr Cys Phe Phe Arg Glu Asp Gly Ser Tyr Glu Glu Ala Leu Val His

100 105 110

Leu Lys Val Ala Ala Leu Gly Ser Asp Pro His Ile Ser Met Gln Val

115 120 125

Gln Glu Asn Gly Glu Ile Cys Leu Glu Cys Thr Ser Val Gly Trp Tyr

130 135 140

Pro Glu Pro Gln Val Gln Trp Arg Thr Ser Lys Gly Glu Lys Phe Pro

145 150 155 160

Ser Thr Ser Glu Ser Arg Asn Pro Asp Glu Glu Gly Leu Phe Thr Val

165 170 175

Ala Ala Ser Val Ile Ile Arg Asp Thr Ser Ala Lys Asn Val Ser Cys

180 185 190

Tyr Ile Gln Asn Leu Leu Leu Gly Gln Glu Lys Lys Val Glu Ile Ser

195 200 205

Ile Pro Ala Ser Ser Leu Pro Arg Asp Lys Thr His Thr Cys Pro Pro

210 215 220

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>4

<211>222

<212>PRT

<213> Artificial sequence

<220>

<223> synthetic polypeptide

<400>4

Ala Pro Phe Asp Val Ile Gly Pro Pro Glu Pro Ile Leu Ala Val Val

1 5 10 15

Gly Glu Asp Ala Glu Leu Pro Cys Arg Leu Ser Pro Asn Ala Ser Ala

20 25 30

Glu His Leu Glu Leu Arg Trp Phe Arg Lys LysVal Ser Pro Ala Val

35 40 45

Leu Val His Arg Asp Gly Arg Glu Gln Glu Ala Glu Gln Met Pro Glu

50 55 60

Tyr Arg Gly Arg Ala Thr Leu Val Gln Asp Gly Ile Ala Lys Gly Arg

65 70 75 80

Val Ala Leu Arg Ile Arg Gly Val Arg Val Ser Asp Asp Gly Glu Tyr

85 90 95

Thr Cys Phe Phe Arg Glu Asp Gly Ser Tyr Glu Glu Ala Leu Val His

100 105 110

Leu Lys Val Ala Ala Leu Gly Ser Asp Pro His Ile Ser Met Gln Val

115 120 125

Gln Glu Asn Gly Glu Ile Cys Leu Glu Cys Thr Ser Val Gly Trp Tyr

130 135 140

Pro Glu Pro Gln Val Gln Trp Arg Thr Ser Lys Gly Glu Lys Phe Pro

145 150 155 160

Ser Thr Ser Glu Ser Arg Asn Pro Asp Glu Glu Gly Leu Phe Thr Val

165 170 175

Ala Ala Ser Val Ile Ile Arg Asp Thr Ser Ala Lys Asn Val Ser Cys

180 185 190

Tyr Ile Gln Asn Leu Leu Leu Gly Gln Glu Lys Lys Val GluIle Ser

195 200 205

Ile Pro Ala Ser Ser Leu Pro Arg His His His His His His

210 215 220

<210>5

<211>524

<212>PRT

<213> mice

<400>5

Met Ala Val Pro Thr Asn Ser Cys Leu Leu Val Cys Leu Leu Thr Leu

1 5 10 15

Thr Val Leu Gln Leu Pro Thr Leu Asp Ser Ala Ala Pro Phe Asp Val

20 25 30

Thr Ala Pro Gln Glu Pro Val Leu Ala Leu Val Gly Ser Asp Ala Glu

35 40 45

Leu Thr Cys Gly Phe Ser Pro Asn Ala Ser Ser Glu Tyr Met Glu Leu

50 55 60

Leu Trp Phe Arg Gln Thr Arg Ser Lys Ala Val Leu Leu Tyr Arg Asp

65 70 75 80

Gly Gln Glu Gln Glu Gly Gln Gln Met Thr Glu Tyr Arg Gly Arg Ala

85 90 95

Thr Leu Ala Thr Ala Gly Leu Leu Asp Gly Arg Ala Thr Leu Leu Ile

100 105 110

Arg Asp Val Arg Val Ser Asp Gln Gly Glu Tyr Arg Cys Leu Phe Lys

115 120 125

Asp Asn Asp Asp Phe Glu Glu Ala Ala Val Tyr Leu Lys Val Ala Ala

130 135 140

Val Gly Ser Asp Pro Gln Ile Ser Met Thr Val Gln Glu Asn Gly Glu

145 150 155 160

Met Glu Leu Glu Cys Thr Ser Ser Gly Trp Tyr Pro Glu Pro Gln Val

165 170 175

Gln Trp Arg Thr Gly Asn Arg Glu Met Leu Pro Ser Thr Ser Glu Ser

180 185 190

Lys Lys His Asn Glu Glu Gly Leu Phe Thr Val Ala Val Ser Met Met

195 200 205

Ile Arg Asp Ser Ser Ile Lys Asn Met Ser Cys Cys Ile Gln Asn Ile

210 215 220

Leu Leu Gly Gln Gly Lys Glu Val Glu Ile Ser Leu Pro Ala Pro Phe

225 230 235 240

Val Pro Arg Leu Thr Pro Trp Ile Val Ala Val Ala Ile Ile Leu Leu

245 250 255

Ala Leu Gly Phe Leu Thr Ile Gly Ser Ile Phe Phe Thr Trp Lys Leu

260 265 270

Tyr LysGlu Arg Ser Ser Leu Arg Lys Lys Glu Phe Gly Ser Lys Glu

275 280 285

Arg Leu Leu Glu Glu Leu Arg Cys Lys Lys Thr Val Leu His Glu Val

290 295 300

Asp Val Thr Leu Asp Pro Asp Thr Ala His Pro His Leu Phe Leu Tyr

305 310 315 320

Glu Asp Ser Lys Ser Val Arg Leu Glu Asp Ser Arg Gln Ile Leu Pro

325 330 335

Asp Arg Pro Glu Arg Phe Asp Ser Trp Pro Cys Val Leu Gly Arg Glu

340 345 350

Thr Phe Thr Ser Gly Arg His Tyr Trp Glu Val Glu Val Gly Asp Arg

355 360 365

Thr Asp Trp Ala Ile Gly Val Cys Arg Glu Asn Val Val Lys Lys Gly

370 375 380

Phe Asp Pro Met Thr Pro Asp Asn Gly Phe Trp Ala Val Glu Leu Tyr

385 390 395 400

Gly Asn Gly Tyr Trp Ala Leu Thr Pro Leu Arg Thr Ser Leu Arg Leu

405 410 415

Ala Gly Pro Pro Arg Arg Val Gly Val Phe Leu Asp Tyr Asp Ala Gly

420 425 430

Asp Ile Ser PheTyr Asn Met Ser Asn Gly Ser Leu Ile Tyr Thr Phe

435 440 445

Pro Ser Ile Ser Phe Ser Gly Pro Leu Arg Pro Phe Phe Cys Leu Trp

450 455 460

Ser Cys Gly Lys Lys Pro Leu Thr Ile Cys Ser Thr Ala Asn Gly Pro

465 470 475 480

Glu Lys Val Thr Val Ile Ala Asn Val Gln Asp Asp Ile Pro Leu Ser

485 490 495

Pro Leu Gly Glu Gly Cys Thr Ser Gly Asp Lys Asp Thr Leu His Ser

500 505 510

Lys Leu Ile Pro Phe Ser Pro Ser Gln Ala Ala Pro

515 520

<210>6

<211>1575

<212>DNA

<213> mice

<400>6

atggcagttc ccaccaactc ctgcctcctg gtctgtctgc tcaccctcac tgtcctacag 60

ctgcccacgc tggattcggc agctcccttc gatgtgaccg cacctcagga gccagtgttg 120

gccctagtgg gctcagatgc cgagctgacc tgtggctttt ccccaaacgc gagctcagaa 180

tacatggagc tgctgtggtt tcgacagacg aggtcgaaag cggtacttct ataccgggat 240

ggccaggagc aggagggcca gcagatgacg gagtaccgcg ggagggcgac gctggcgaca 300

gccgggcttc tagacggccg cgctactctg ctgatccgag atgtcagggt ctcagaccag 360

ggggagtacc ggtgcctttt caaagacaac gacgacttcg aggaggccgc cgtatacctc 420

aaagtggctg ctgtgggttc agatcctcaa atcagtatga cggttcaaga gaatggagaa 480

atggagctgg agtgcacctc ctctggatgg tacccagagc ctcaggtgca gtggagaaca 540

ggcaacagag agatgctacc atccacgtca gagtccaaga agcataatga ggaaggcctg 600

ttcactgtgg cagtttcaat gatgatcaga gacagctcca taaagaacat gtcctgctgc 660

atccagaata tcctccttgg ccaggggaag gaagtagaga tctccttacc agctcccttc 720

gtgccaaggc tgactccctg gatagtagct gtggctatca tcttactggc cttaggattt 780

ctcaccattg ggtccatatt tttcacttgg aaactataca aggaaagatc cagtctgcgg 840

aagaaggaat ttggctctaa agagagactt ctggaagaac tcagatgcaa aaagactgta 900

ctgcatgaag ttgacgtgac tctggatcca gacacagccc acccccacct cttcctgtat 960

gaagattcaa agtcagttcg attggaagat tcacgtcaga tcctgcctga tagaccagag 1020

agatttgact cctggccctg tgtgttgggc cgtgagacct ttacttcagg gagacattac 1080

tgggaggtgg aggtgggaga tagaactgac tgggccattg gtgtgtgtag ggagaatgtg 1140

gtgaagaaag ggtttgaccc catgactcct gataatgggt tctgggctgt ggagttgtat 1200

ggaaatgggt actgggccct caccccactc aggacctctc tccgattagc agggccccct 1260

cgcagagttg gggtttttct ggactatgac gcaggagaca tttccttcta caacatgagt 1320

aacggatctc ttatctatac tttccctagc atctctttct ctggccccct ccgtcccttc 1380

ttttgtctgt ggtcctgtgg taaaaagccc ctgaccatct gttcaactgc caatgggcct 1440

gagaaagtca cagtcattgc taatgtccag gacgacattc ccttgtcccc gctgggggaa 1500

ggctgtactt ctggagacaa agacactctc cattctaaac tgatcccgtt ctcacctagc 1560

caagcggcac cataa 1575

<210>7

<211>135

<212>PRT

<213> Intelligent people

<400>7

Met Ala Cys Gly Leu Val Ala Ser Asn Leu Asn Leu Lys Pro Gly Glu

1 5 10 15

Cys Leu Arg Val Arg Gly Glu Val Ala Pro Asp Ala Lys Ser Phe Val

20 25 30

Leu Asn Leu Gly Lys Asp Ser Asn Asn Leu Cys Leu His Phe Asn Pro

35 40 45

Arg Phe Asn Ala His Gly Asp Ala Asn Thr Ile Val Cys Asn Ser Lys

50 55 60

Asp Gly Gly Ala Trp Gly Thr Glu Gln Arg Glu Ala Val Phe Pro Phe

65 70 75 80

Gln Pro Gly Ser Val Ala Glu Val Cys Ile Thr Phe Asp Gln Ala Asn

85 90 95

Leu Thr Val Lys Leu Pro Asp Gly Tyr Glu Phe Lys Phe Pro Asn Arg

100 105 110

Leu Asn Leu Glu Ala Ile Asn Tyr Met Ala Ala Asp Gly Asp Phe Lys

115 120 125

Ile Lys Cys Val Ala Phe Asp

130 135

<210>8

<211>408

<212>DNA

<213> Intelligent people

<400>8

atggcttgtg gtctggtcgc cagcaacctg aatctcaaac ctggagagtg ccttcgagtg 60

cgaggcgagg tggctcctga cgctaagagc ttcgtgctga acctgggcaa agacagcaac 120

aacctgtgcc tgcacttcaa ccctcgcttc aacgcccacg gcgacgccaa caccatcgtg 180

tgcaacagca aggacggcgg ggcctggggg accgagcagc gggaggctgt ctttcccttc 240

cagcctggaa gtgttgcaga ggtgtgcatc accttcgacc aggccaacct gaccgtcaag 300

ctgccagatg gatacgaatt caagttcccc aaccgcctca acctggaggc catcaactac 360

atggcagctg acggtgactt caagatcaaa tgtgtggcct ttgactga 408

<210>9

<211>246

<212>PRT

<213> Intelligent people

<400>9

Met Ala Phe Ser Gly Ser Gln Ala Pro Tyr Leu Ser Pro Ala Val Pro

1 5 10 15

Phe Ser Gly Thr Ile Gln Gly Gly Leu Gln Asp Gly Leu Gln Ile Thr

20 25 30

Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg Phe Ala Val Asn

35 40 45

Phe Gln Thr Gly Phe Ser Gly Asn Asp Ile Ala Phe His Phe Asn Pro

50 55 60

Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr Arg Gln Asn Gly

65 70 75 80

Ser Trp Gly Pro Glu Glu Arg Lys Thr His Met Pro Phe Gln Lys Gly

85 90 95

Met Pro Phe Asp Leu Cys Phe Leu Val Gln Ser Ser Asp Phe Lys Val

100 105 110

Met Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His Arg Val Pro Phe

115 120 125

His Arg Val Asp Thr Ile Ser Val Asn Gly Ser Val Gln Leu Ser Tyr

130 135 140

Ile Ser Phe Gln Pro Pro Gly Val Trp Pro Ala Asn Pro Ala Pro Ile

145 150 155 160

Thr Gln Thr Val Ile His Thr Val Gln Ser Ala Pro Gly Gln Met Phe

165 170 175

Ser Thr Pro Ala Ile Pro Pro Met Met Tyr Pro His Pro Ala Tyr Pro

180 185190

Met Pro Phe Ile Thr Thr Ile Leu Gly Gly Leu Tyr Pro Ser Lys Ser

195 200 205

Ile Leu Leu Ser Gly Thr Val Leu Pro Ser Ala Gln Arg Cys Gly Ser

210 215 220

Cys Val Lys Leu Thr Ala Ser Arg Trp Pro Trp Met Val Ser Thr Cys

225 230 235 240

Leu Asn Thr Thr Ile Ala

245

<210>10

<211>971

<212>DNA

<213> Intelligent people

<400>10

atggccttca gcggttccca ggctccctac ctgagtccag ctgtcccctt ttctgggact 60

attcaaggag gtctccagga cggacttcag atcactgtca atgggaccgt tctcagctcc 120

agtggaacca ggtttgctgt gaactttcag actggcttca gtggaaatga cattgccttc 180

cacttcaacc ctcggtttga agatggaggg tacgtggtgt gcaacacgag gcagaacgga 240

agctgggggc ccgaggagag gaagacacac atgcctttcc agaaggggat gccctttgac 300

ctctgcttcc tggtgcagag ctcagatttc aaggtgatgg tgaacgggat cctcttcgtg 360

cagtacttcc accgcgtgcc cttccaccgt gtggacacca tctccgtcaa tggctctgtg 420

cagctgtcct acatcagctt ccagcctccc ggcgtgtggc ctgccaaccc ggctcccatt 480

acccagacag tcatccacac agtgcagagc gcccctggac agatgttctc tactcccgcc 540

atcccactat gatgtacccc caccccgcct atccgatgcc tttcatcacc accattctgg 600

gagggctgta cccatccaag tccatcctcc tgtcaggcac tgtcctgccc agtgctcaga 660

ggttccacat caacctgtgc tctgggaacc acatcgcctt ccacctgaac ccccgttttg 720

atgagaatgc tgtggtccgc aacacccaga tcgacaactc ctgggggtct gaggagcgaa 780

gtctgccccg aaaaatgccc ttcgtccgtg gccagagctt ctcagtgtgg atcttgtgtg 840

aagctcactg cctcaaggtg gccgtggatg gtcagcacct gtttgaatac taccatcgcc 900

tgaggaacct gcccaccatc aacagactgg aagtgggggg cgacatccag ctgacccatg 960

tgcagacata g 971

<210>11

<211>926

<212>PRT

<213> Intelligent people

<400>11

Met Asp Met Phe Pro Leu Thr Trp Val Phe Leu Ala Leu Tyr Phe Ser

1 5 10 15

Arg His Gln Val Arg Gly Gln Pro Asp Pro Pro Cys Gly Gly Arg Leu

20 25 30

Asn Ser Lys Asp Ala Gly Tyr Ile Thr Ser Pro Gly Tyr Pro Gln Asp

35 40 45

Tyr Pro Ser His Gln Asn Cys Glu Trp Ile Val Tyr Ala Pro Glu Pro

50 55 60

Asn Gln Lys Ile Val Leu Asn Phe Asn Pro His Phe Glu Ile Glu Lys

65 70 75 80

His Asp Cys Lys Tyr Asp Phe Ile Glu Ile Arg Asp Gly Asp Ser Glu

85 90 95

Ser Ala Asp Leu Leu Gly Lys His Cys Gly Asn Ile Ala Pro Pro Thr

100 105 110

Ile Ile Ser Ser Gly Ser Met Leu Tyr Ile Lys Phe Thr Ser Asp Tyr

115 120 125

Ala Arg Gln Gly Ala Gly Phe Ser Leu Arg Tyr Glu Ile Phe Lys Thr

130 135 140

Gly Ser Glu Asp Cys Ser Lys Asn Phe Thr Ser Pro Asn Gly Thr Ile

145 150 155 160

Glu Ser Pro Gly Phe Pro Glu Lys Tyr Pro His Asn Leu Asp Cys Thr

165 170 175

Phe Thr Ile Leu Ala Lys Pro Lys Met Glu Ile Ile Leu Gln Phe Leu

180 185 190

Ile Phe Asp Leu Glu His Asp Pro Leu Gln Val Gly Glu Gly Asp Cys

195 200 205

Lys Tyr Asp Trp Leu Asp Ile Trp Asp Gly Ile Pro His Val Gly Pro

210 215 220

Leu Ile Gly Lys Tyr Cys Gly Thr Lys Thr Pro Ser Glu Leu Arg Ser

225 230 235 240

Ser Thr Gly Ile Leu Ser Leu Thr Phe His Thr Asp Met Ala Val Ala

245 250 255

Lys Asp Gly Phe Ser Ala Arg Tyr Tyr Leu Val His Gln Glu Pro Leu

260 265 270

Glu Asn Phe Gln Cys Asn Val Pro Leu Gly Met Glu Ser Gly Arg Ile

275 280 285

Ala Asn Glu Gln Ile Ser Ala Ser Ser Thr Tyr Ser Asp Gly Arg Trp

290 295 300

Thr Pro Gln Gln Ser Arg Leu His Gly Asp Asp Asn Gly Trp Thr Pro

305 310 315 320

Asn Leu Asp Ser Asn Lys Glu Tyr Leu Gln Val Asp Leu Arg Phe Leu

325 330 335

Thr Met Leu Thr Ala Ile Ala Thr Gln Gly Ala Ile Ser Arg Glu Thr

340 345 350

Gln Asn Gly Tyr Tyr Val Lys Ser Tyr Lys Leu Glu Val Ser Thr Asn

355 360 365

Gly Glu Asp Trp Met Val Tyr Arg His Gly Lys Asn His Lys Val Phe

370 375 380

Gln Ala Asn Asn Asp Ala Thr Glu Val Val Leu Asn Lys Leu His Ala

385 390 395 400

Pro Leu Leu Thr Arg Phe Val Arg Ile Arg Pro Gln Thr Trp His Ser

405 410 415

Gly Ile Ala Leu Arg Leu Glu Leu Phe Gly Cys Arg Val Thr Asp Ala

420 425 430

Pro Cys Ser Asn Met Leu Gly Met Leu Ser Gly Leu Ile Ala Asp Ser

435 440 445

Gln Ile Ser Ala Ser Ser Thr Gln Glu Tyr Leu Trp Ser Pro Ser Ala

450 455 460

Ala Arg Leu Val Ser Ser Arg Ser Gly Trp Phe Pro Arg Ile Pro Gln

465 470 475 480

Ala Gln Pro Gly Glu Glu Trp Leu Gln Val Asp Leu Gly Thr Pro Lys

485 490 495

Thr Val Lys Gly Val Ile Ile Gln Gly Ala Arg Gly Gly Asp Ser Ile

500 505 510

Thr Ala Val Glu Ala Arg Ala Phe Val Arg Lys Phe Lys Val Ser Tyr

515 520 525

Ser Leu Asn Gly Lys Asp Trp Glu Tyr Ile Gln Asp Pro Arg Thr Gln

530 535 540

Gln Pro Lys Leu Phe Glu Gly Asn Met His Tyr Asp Thr Pro Asp Ile

545 550 555 560

Arg Arg Phe Asp Pro Ile Pro Ala Gln Tyr Val Arg Val Tyr Pro Glu

565 570 575

Arg Trp Ser Pro Ala Gly Ile Gly Met Arg Leu Glu Val Leu Gly Cys

580 585 590

Asp Trp Thr Asp Ser Lys Pro Thr Val Glu Thr Leu Gly Pro Thr Val

595 600 605

Lys Ser Glu Glu Thr Thr Thr Pro Tyr Pro Thr Glu Glu Glu Ala Thr

610 615 620

Glu Cys Gly Glu Asn Cys Ser Phe Glu Asp Asp Lys Asp Leu Gln Leu

625 630 635 640

Pro Ser Gly Phe Asn Cys Asn Phe Asp Phe Leu Glu Glu Pro Cys Gly

645 650 655

Trp Met Tyr Asp His Ala Lys Trp Leu Arg Thr Thr Trp Ala Ser Ser

660 665 670

Ser Ser Pro Asn Asp Arg Thr Phe Pro Asp Asp Arg Asn Phe Leu Arg

675 680 685

Leu Gln Ser Asp Ser Gln Arg Glu Gly Gln Tyr Ala Arg Leu Ile Ser

690 695 700

Pro Pro Val His Leu Pro Arg Ser Pro Val Cys Met Glu Phe Gln Tyr

705 710 715 720

Gln Ala Thr Gly Gly Arg Gly Val Ala Leu Gln Val Val Arg Glu Ala

725 730 735

Ser Gln Glu Ser Lys Leu Leu Trp Val Ile Arg Glu Asp Gln Gly Gly

740 745 750

Glu Trp Lys His Gly Arg Ile Ile Leu Pro Ser Tyr Asp Met Glu Tyr

755 760 765

Gln Ile Val Phe Glu Gly Val Ile Gly Lys Gly Arg Ser Gly Glu Ile

770 775 780

Ala Ile Asp Asp Ile Arg Ile Ser Thr Asp Val Pro Leu Glu Asn Cys

785 790 795 800

Met Glu Pro Ile Ser Ala Phe Ala Val Asp Ile Pro Glu Ile His Glu

805 810 815

Arg Glu Gly Tyr Glu Asp Glu Ile Asp Asp Glu Tyr Glu Val Asp Trp

820 825 830

Ser Asn Ser Ser Ser Ala Thr Ser Gly Ser Gly Ala Pro Ser Thr Asp

835 840 845

Lys Glu Lys Ser Trp Leu Tyr Thr Leu Asp Pro Ile Leu Ile Thr Ile

850 855 860

Ile Ala Met Ser Ser Leu Gly Val Leu Leu Gly Ala Thr Cys Ala Gly

865870 875 880

Leu Leu Leu Tyr Cys Thr Cys Ser Tyr Ser Gly Leu Ser Ser Arg Ser

885 890 895

Cys Thr Thr Leu Glu Asn Tyr Asn Phe Glu Leu Tyr Asp Gly Leu Lys

900 905 910

His Lys Val Lys Met Asn His Gln Lys Cys Cys Ser Glu Ala

915 920 925

<210>12

<211>2775

<212>DNA

<213> Intelligent people

<400>12

atgtttcctc tcacctgggt tttcttagcc ctctactttt caagacacca agtgagaggc 60

caaccagacc caccgtgcgg aggtcgtttg aattccaaag atgctggcta tatcacctct 120

cccggttacc cccaggacta cccctcccac cagaactgcg agtggattgt ttacgccccc 180

gaacccaacc agaagattgt cctcaacttc aaccctcact ttgaaatcga gaagcacgac 240

tgcaagtatg actttatcga gattcgggat ggggacagtg aatccgcaga cctcctgggc 300

aaacactgtg ggaacatcgc cccgcccacc atcatctcct cgggctccat gctctacatc 360

aagttcacct ccgactacgc ccggcagggg gcaggcttct ctctgcgcta cgagatcttc 420

aagacaggct ctgaagattg ctcaaaaaac ttcacaagcc ccaacgggac catcgaatct 480

cctgggtttc ctgagaagta tccacacaac ttggactgca cctttaccat cctggccaaa 540

cccaagatgg agatcatcct gcagttcctg atctttgacctggagcatga ccctttgcag 600

gtgggagagg gggactgcaa gtacgattgg ctggacatct gggatggcat tccacatgtt 660

ggccccctga ttggcaagta ctgtgggacc aaaacaccct ctgaacttcg ttcatcgacg 720

gggatcctct ccctgacctt tcacacggac atggcggtgg ccaaggatgg cttctctgcg 780

cgttactacc tggtccacca agagccacta gagaactttc agtgcaatgt tcctctgggc 840

atggagtctg gccggattgc taatgaacag atcagtgcct catctaccta ctctgatggg 900

aggtggaccc ctcaacaaag ccggctccat ggtgatgaca atggctggac ccccaacttg 960

gattccaaca aggagtatct ccaggtggac ctgcgctttt taaccatgct cacggccatc 1020

gcaacacagg gagcgatttc cagggaaaca cagaatggct actatgtcaa atcctacaag 1080

ctggaagtca gcactaatgg agaggactgg atggtgtacc ggcatggcaa aaaccacaag 1140

gtatttcaag ccaacaacga tgcaactgag gtggttctga acaagctcca cgctccactg 1200

ctgacaaggt ttgttagaat ccgccctcag acctggcact caggtatcgc cctccggctg 1260

gagctcttcg gctgccgggt cacagatgct ccctgctcca acatgctggg gatgctctca 1320

ggcctcattg cagactccca gatctccgcc tcttccaccc aggaatacct ctggagcccc 1380

agtgcagccc gcctggtcag cagccgctcg ggctggttcc ctcgaatccc tcaggcccag 1440

cccggtgagg agtggcttca ggtagatctg ggaacaccca agacagtgaa aggtgtcatc 1500

atccagggag cccgcggagg agacagtatc actgctgtgg aagccagagc atttgtgcgc 1560

aagttcaaag tctcctacag cctaaacggc aaggactggg aatacattca ggaccccagg 1620

acccagcagc caaagctgtt cgaagggaac atgcactatg acacccctga catccgaagg1680

tttgacccca ttccggcaca gtatgtgcgg gtatacccgg agaggtggtc gccggcgggg 1740

attgggatgc ggctggaggt gctgggctgt gactggacag actccaagcc cacggtagag 1800

acgctgggac ccactgtgaa gagcgaagag acaaccaccc cctaccccac cgaagaggag 1860

gccacagagt gtggggagaa ctgcagcttt gaggatgaca aagatttgca gctcccttcg 1920

ggattcaatt gcaacttcga tttcctcgag gagccctgtg gttggatgta tgaccatgcc 1980

aagtggctcc ggaccacctg ggccagcagc tccagcccaa acgaccggac gtttccagat 2040

gacaggaatt tcttgcggct gcagagtgac agccagagag agggccagta tgcccggctc 2100

atcagccccc ctgtccacct gccccgaagc ccggtgtgca tggagttcca gtaccaggcc 2160

acgggcggcc gcggggtggc gctgcaggtg gtgcgggaag ccagccagga gagcaagttg 2220

ctgtgggtca tccgtgagga ccagggcggc gagtggaagc acgggcggat catcctgccc 2280

agctacgaca tggagtacca gattgtgttc gagggagtga tagggaaagg acgttccgga 2340

gagattgcca ttgatgacat tcggataagc actgatgtcc cactggagaa ctgcatggaa 2400

cccatctcgg cttttgcagt ggacatccca gaaatacatg agagagaagg atatgaagat 2460

gaaattgatg atgaatacga ggtggactgg agcaattctt cttctgcaac ctcagggtct 2520

ggcgccccct cgaccgacaa agaaaagagc tggctgtaca ccctggatcc catcctcatc 2580

accatcatcg ccatgagctc actgggcgtc ctcctggggg ccacctgtgc aggcctcctg 2640

ctctactgca cctgttccta ctcgggcctg agctcccgaa gctgcaccac actggagaac 2700

tacaacttcg agctctacga tggccttaag cacaaggtca agatgaacca ccaaaagtgc 2760

tgctccgagg catga 2775

<210>13

<211>289

<212>PRT

<213> Intelligent people

<400>13

Met Lys Thr Leu Pro Ala Met Leu Gly Thr Gly Lys Leu Phe Trp Val

1 5 10 15

Phe Phe Leu Ile Pro Tyr Leu Asp Ile Trp Asn Ile His Gly Lys Glu

20 25 30

Ser Cys Asp Val Gln Leu Tyr Ile Lys Arg Gln Ser Glu His Ser Ile

35 40 45

Leu Ala Gly Asp Pro Phe Glu Leu Glu Cys Pro Val Lys Tyr Cys Ala

50 55 60

Asn Arg Pro His Val Thr Trp Cys Lys Leu Asn Gly Thr Thr Cys Val

65 70 75 80

Lys Leu Glu Asp Arg Gln Thr Ser Trp Lys Glu Glu Lys Asn Ile Ser

85 90 95

Phe Phe Ile Leu His Phe Glu Pro Val Leu Pro Asn Asp Asn Gly Ser

100 105 110

Tyr Arg Cys Ser Ala Asn Phe Gln Ser Asn Leu Ile Glu Ser His Ser

115 120 125

Thr Thr Leu Tyr Val Thr Asp Val Lys Ser Ala Ser Glu Arg Pro Ser

130 135 140

Lys Asp Glu Met Ala Ser Arg Pro Trp Leu Leu Tyr Arg Leu Leu Pro

145 150 155 160

Leu Gly Gly Leu Pro Leu Leu Ile Thr Thr Cys Phe Cys Leu Phe Cys

165 170 175

Cys Leu Arg Arg His Gln Gly Lys Gln Asn Glu Leu Ser Asp Thr Ala

180 185 190

Gly Arg Glu Ile Asn Leu Val Asp Ala His Leu Lys Ser Glu Gln Thr

195 200 205

Glu Ala Ser Thr Arg Gln Asn Ser Gln Val Leu Leu Ser Glu Thr Gly

210 215 220

Ile Tyr Asp Asn Asp Pro Asp Leu Cys Phe Arg Met Gln Glu Gly Ser

225 230 235 240

Glu Val Tyr Ser Asn Pro Cys Leu Glu Glu Asn Lys Pro Gly Ile Val

245 250 255

Tyr Ala Ser Leu Asn His Ser Val Ile Gly Pro Asn Ser Arg Leu Ala

260 265 270

Arg Asn Val Lys Glu Ala Pro Thr Glu Tyr Ala Ser Ile Cys Val Arg

275 280 285

Ser

<210>14

<211>726

<212>DNA

<213> Intelligent people

<400>14

atgaagacat tgcctgccat gcttggaact gggaaattat tttgggtctt cttcttaatc 60

ccatatctgg acatctggaa catccatggg aaagaatcat gtgatgtaca gctttatata 120

aagagacaat ctgaacactc catcttagca ggagatccct ttgaactaga atgccctgtg 180

aaatactgtg ctaacaggcc tcatgtgact tggtgcaagc tcaatggaac aacatgtgta 240

aaacttgaag atagacaaac aagttggaag gaagagaaga acatttcatt tttcattcta 300

cattttgaac cagtgcttcc taatgacaat gggtcatacc gctgttctgc aaattttcag 360

tctaatctca ttgaaagcca ctcaacaact ctttatgtga caggaaagca aaatgaactc 420

tctgacacag caggaaggga aattaacctg gttgatgctc accttaagag tgagcaaaca 480

gaagcaagca ccaggcaaaa ttcccaagta ctgctatcag aaactggaat ttatgataat 540

gaccctgacc tttgtttcag gatgcaggaa gggtctgaag tttattctaa tccatgcctg 600

gaagaaaaca aaccaggcat tgtttatgct tccctgaacc attctgtcat tggaccgaac 660

tcaagactgg caagaaatgt aaaagaagca ccaacagaat atgcatccat atgtgtgagg 720

agttaa 726

Claims

1. A molecule comprising an antigen-binding fragment that immunospecifically binds to BTN1a1, wherein said molecule inhibits the binding of BTN1a1 to BTN1a1 ligand, said BTN1a1 ligand being selected from the group consisting of galectin-1 (GAL-1), galectin-9 (GAL-9), NRP-2(NrP-2), and B-and T-lymphokine attenuated protein (BTLA).

2. The molecule of claim 1, wherein the antigen-binding fragment immunospecifically binds to BTN1a1, and the molecule inhibits binding of BTN1a1 to GAL-1.

3. The molecule of claim 1 or 2, wherein the antigen-binding fragment immunospecifically binds to BTN1a1, and the molecule inhibits binding of BTN1a1 to GAL-9.

4. The molecule of any one of claims 1-3, wherein the antigen-binding fragment immunospecifically binds BTN1A1, and the molecule inhibits binding of BTN1A1 to NRP-2.

5. The molecule of any one of claims 1-4, wherein the antigen-binding fragment immunospecifically binds BTN1A1, and the molecule inhibits binding of BTN1A1 to BTLA.

6. The molecule of any one of claims 1-5, wherein the antigen-binding fragment immunospecifically binds to BTN1A1, and the molecule inhibits BTN1A1 from binding two or more BN1A1 ligands selected from GAL-1, GAL-9, NRP-2, or BTLA.

7. The molecule of any one of claims 1-7, wherein the antigen-binding fragment immunospecifically binds to the extracellular domain (ECD) of BTN1a 1.

8. A molecule comprising an antigen-binding fragment that immunospecifically binds to a BTN1a1 ligand, said BTN1a1 ligand being selected from the group consisting of GAL-1, GAL-9, NRP-2, and BTLA, said molecule inhibiting the binding of BTN1a1 ligand to BTN1a 1.

9. The molecule of claim 8, wherein the antigen-binding fragment immunospecifically binds GAL-1, and the molecule inhibits binding of GAL-1 to BTN1a 1.

10. The molecule of claim 8, wherein the antigen-binding fragment immunospecifically binds GAL-9, and the molecule inhibits binding of GAL-9 to BTN1a 1.

11. The molecule of claim 8, wherein the antigen-binding fragment immunospecifically binds to NRP-2, and the molecule inhibits binding of NRP-2 to BTN1a 1.

12. The molecule of claim 8, wherein the antigen-binding fragment immunospecifically binds BTLA and the molecule inhibits binding of BTLA to BTN1A 1.

13. The molecule of any one of claims 1-12, wherein the molecule completely inhibits binding of BTN1a1 ligand to BTN1a 1.

14. The molecule of any one of claims 1-12, wherein the molecule inhibits at least 1%, at least 3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the binding of BTN1a1 to BTN1a1 ligand.

15. The molecule of any one of claims 1-14, wherein the molecule modulates activity or signaling of BTN1a1, or of a BTN1a1 complex with a BTN1a1 ligand, such as GAL-1, GAL-9, NRP-2, or BTLA.

16. The molecule of any one of claims 1-15, wherein the molecule modulates T cell activity.

17. The molecule of claim 16, wherein the T cell is a CD8+ T cell.

18. The molecule of claim 16 or 17, wherein the molecule increases T cell activation or T cell proliferation.

19. The molecule of any one of claims 16-18, wherein the molecule inhibits T cell apoptosis.

20. The molecule of any one of claims 1-19, wherein the antigen-binding fragment preferentially binds glycosylated BTN1a1 relative to non-monomeric BTN1a1 glycosylated BTN1a 1.

21. The molecule of any one of claims 1-20, wherein the antigen-binding fragment preferentially binds to dimeric BTN1a1 relative to monomeric BTN1a 1.

22. The molecule of any one of claims 1-21, wherein the antigen-binding fragment has a dissociation constant (K) of no greater than 1 μ M_D) Immunospecifically binds to BTN1a1 or BTN1a1 ligand.

23. The molecule of any one of claims 1-22, wherein the antigen-binding fragment has a dissociation constant (K) of no greater than 500nM, no greater than 400nM, no greater than 300nM, no greater than 200nM, no greater than 100nM, no greater than 50nM, no greater than 10nM, or no greater than 5nM_D) Immunospecifically binds to BTN1a1 or BTN1a1 ligand.

24. The molecule of any one of claims 1-23, wherein the antigen-binding fragment immunospecifically binds to K of BTN1a1 or BTN1a1 ligand_DK less than or equal to BTN1A1-GAL-1 interaction, BTN1A1-GAL-9 interaction, BTN1A1-NRP2 interaction, or BTN1A1-BTLA interaction_D。

25. The molecule of any one of claims 1-24, wherein the antigen-binding fragment immunospecifically binds to K of BTN1a1 or BTN1a1 ligand_DK compared to BTN1A1-GAL-1-1 interaction, BTN1A1-GAL-9 interaction, BTN1A1-NRP2 interaction, or BTN1A1-BTLA interaction_DAt least 2 times lower, at least 5 times lower, at least 10 times lower, at least 15 times lower, at least 20 times lower, at least 25 times lower, at least 30 times lower, at least 40 times lower or at least 50 times lower.

26. The molecule of any one of claims 1-25, wherein the molecule has an IC50 of no greater than 1 μ Μ that inhibits binding of BTN1a1 ligand to BTN1a 1.

27. The molecule of any one of claims 1-26, wherein the molecule has an IC50 of no greater than 500nM, no greater than 400nM, no greater than 300nM, no greater than 200nM, no greater than 100nM, no greater than 50nM, no greater than 10nM, or no greater than 5nM that inhibits binding of a BTN1a1 ligand to BTN1a 1.

28. The molecule of any one of claims 1-27, wherein inhibition of BTN1a1 binding, BTN1a1 ligand binding, or BTN1a1 ligand binding to BTN1a1 binding is assayed by co-immunoprecipitation (co-IP), Surface Plasmon Resonance (SPR) assay, b-galactosidase complementation, or biolayer interferometry (BLI).

29. The molecule of any one of claims 1-28, wherein the molecule is an antibody.

30. A molecule according to claim 29, wherein the antibody is a monoclonal antibody.

31. A molecule according to claim 29 or 30, wherein the antibody is a human or humanized antibody.

32. The molecule of any one of claims 29-31, wherein the antibody is an IgG, IgM, or IgA.

33. The molecule of any one of claims 1-32, wherein the molecule is a Fab ', F (ab ') 2, F (ab ') 3, monovalent scFv, bivalent scFv, or single domain antibody.

34. The molecule of any one of claims 1-33, wherein the molecule is recombinantly produced.

35. A pharmaceutical composition comprising the molecule of any one of claims 1-34 and a pharmaceutically acceptable carrier.

36. The medicament of claim 35, formulated for parenteral administration.

37. A method of activating a T cell, comprising contacting a T cell with an effective amount of the molecule of any one of claims 1-34, wherein the T cell is activated by inhibiting binding of a BTN1a1 ligand to BTN1a 1.

38. A method of inhibiting binding of a BTN1a1 ligand to BTN1a1 expressed by a T cell, comprising contacting the T cell with an effective amount of the molecule of any one of claims 1-34, thereby inhibiting binding of the BTN1a1 ligand to the T cell.

39. The method of claim 37 or 38, wherein the T cell is a CD8+ cell.

40. The method of any one of claims 37-39, wherein T cell activation comprises (i) increasing T cell proliferation, (ii) decreasing T cell apoptosis, or (iii) increasing cytokine production.

41. The method of claim 40, wherein the cytokine is IFN- γ or IL-2.

42. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the molecule of any one of claims 1-34 or the pharmaceutical composition of claim 35 or 36, wherein the molecule inhibits binding of BTN1a1 ligand to BTN1a1 in the subject.

43. A method of inhibiting binding of BTN1a1 ligand to BTN1a1 by administering to a subject a therapeutically effective amount of the molecule of any one of claims 1-34 or the pharmaceutical composition of claim 35 or 36, wherein the molecule inhibits binding of BTN1a1 ligand to BTN1a1 in the subject.

44. The method of claim 42 or 43, said administering comprising parenteral administration of said molecule or said pharmaceutical composition.

45. The method of any one of claims 42-44, further comprising administering high dose radiation therapy to the patient.

46. The method of any one of claims 42-45, wherein the cancer is selected from lung cancer, prostate cancer, pancreatic cancer, ovarian cancer, liver cancer, head and neck cancer, breast cancer, and gastric cancer.

47. The method of any one of claims 42-46, wherein the cancer is a lung cancer.

48. The method of any one of claims 42-47, wherein the lung cancer is non-small cell lung cancer (NSCLC).

49. The method of any one of claims 42-48, wherein the NSCLC is squamous NSCLC.

50. The method of any one of claims 42-49, wherein the cancer is an anti-PD-1 therapy or an anti-PD-L1 therapy resistant or refractory cancer.

51. The method of claim 50, wherein the cancer is a breast cancer or a lung cancer.

52. The method of claim 51, wherein the cancer is breast cancer or Lewis lung cancer.

53. The method of any one of claims 1-52, wherein the molecule is not STC810 as described in International application PCT/US16/64436 or does not comprise a CDR or VH or VL amino acid sequence of STC 810.